Tumor suppressor gene p33ING2

ABSTRACT

The invention provides isolated nucleic acid and amino acid sequences of novel human tumor suppressors, antibodies to such tumor suppressors, methods of detecting such nucleic acids and proteins, methods of screening for modulators of tumor suppressors, and methods of diagnosing and treating tumors with such nucleic acids and proteins.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to provisional application U.S.S. No.60/121,891, filed Feb. 26, 1999, the disclosure of which is hereinincorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

This invention relates to isolated nucleic acid and amino acid sequencesof novel human tumor suppressors, antibodies to such tumor suppressors,methods of detecting such nucleic acids and proteins, methods ofscreening for modulators of tumor suppressors, and methods of diagnosingand treating tumors with such nucleic acids and proteins.

BACKGROUND OF THE INVENTION

Certain tumors, benign, premalignant, and malignant, are known to havegenetic components. Some of these tumors are caused by mutations orinactivation of “tumor suppressor” genes. In normal cells, the tumorsuppressor genes are involved in the regulation of cell growth andproliferation and in the control of cellular aging, anchorage dependenceand apoptosis. When the tumor suppressor genes are mutated orinactivated, cells are transformed and become immortalized ortumorigenic. These transformed cells can be reverted back to the normalphenotype (i.e., the cell growth rate is suppressed) by introducing thewildtype suppressor genes.

The first tumor suppressor gene identified was the nuclearphosphoprotein, retinoblastoma gene (Rb). Retinoblastoma is a malignanttumor of the sensory layer of the retina, and often occurs bilaterallyduring childhood. Retinoblastoma exhibits a familial tendency, but itcan be acquired. Mutations in the Rb gene and inactivation of itsproduct have been shown to be involved in other tumors, such as bladder,breast, small cell lung carcinomas, osteosarcomas, and soft tissuesarcomas. It was demonstrated that reconstitution of Rb-deficient tumorcells with the wildtype Rb leads to the suppression of growth rate ortumorigenicity (Huang et al., Science 242:1563-1566 (1988)). This resultprovides direct evidence that Rb protein is a tumor suppressor.

Another well-characterized tumor suppressor is the gene for the nuclearphosphoprotein, p53. More than half of all human cancers are associatedwith mutations in the tumor suppressor gene p53 (see, e.g., Hollstein etal., Science 253:49-53 (1991); Caron de Fronmentel & Soussi, GenesChromosom. Cancer 4: 1-15; Harris & Hollstein, N. Engl. J. Med.329:1318-1327 (1993); Greenblatt et al., Cancer Res. 54:4855-4878(1994)). Mutations in p53 often appear to be a critical step in thepathogenesis and progression of tumors. For example, missense mutationsof p53 occur in tumors of the colon, lung, breast, ovary, bladder, andseveral other organs. Alternatively, inactivation of the wildtype p53proteins in cells can cause tumors. For example, certain strains ofhuman papillomavirus (HPV) are known to interfere with the p53 proteinfunction, because the virus produces a protein, E6, which promotes thedegradation of the p53 protein.

Recently, another tumor suppressor gene, p33ING1, has been identified.p33ING1 directly cooperates with tumor suppressor gene p53 in growthregulation (Garkavtsev et al., Nature Genetics 14:415-420 (1996);Garkavtsev et al., Nature 391:295-298 (1998); U.S. Pat. No. 5,986,078,all of which are herein incorporated by reference). Neither of p53 orp33ING1 can alone cause growth inhibition when the other one issuppressed (Garkavtsev et al. (1998), supra). According toimmunoprecipitation studies, p33ING1 proteins modulate the p53 activitythrough physical interaction. It has been also reported that someneuroblastoma cells have a mutation of the p33ING1 gene, and some breastcancer cell lines exhibit reduced expression of p33ING1 (Garkavtsev etal. (1996), supra).

Cancer remains a major public concern. Although epidemiological andcytogenetic studies demonstrated that a number of recessive geneticmutations are involved in various cancers, only a limited number oftumor suppressors have been identified. Therefore, there is a need toidentify and isolate other tumor suppressor genes. The identificationand isolation of new tumor suppressor genes would assist the diagnosis,prevention, and treatment of tumors and cancers.

SUMMARY OF THE INVENTION

The present invention thus provides for the first time nucleic acid andamino acid sequences of a new tumor suppressor gene called p33ING2, aswell as antibodies to p33ING2, methods of detecting such nucleic acidsand proteins, methods of screening for modulators of p33ING2, andmethods of diagnosing and treating tumors. P33ING2 nucleic acids andproteins are tumor suppressors that play a key role in regulation ofcell proliferation and tumor suppression.

In one aspect, the present invention provides an isolated nucleic acidencoding a tumor suppressor polypeptide p33ING2, wherein the polypeptidehas greater than 70% amino acid sequence identity to a polypeptidecomprising an amino acid sequence of SEQ ID NO:1. In one embodiment, thenucleic acid encodes a polypeptide that selectively binds to polyclonalantibodies generated against a polypeptide comprising an amino acidsequence of SEQ ID NO:1. In another embodiment, the nucleic acid encodesa polypeptide comprising an amino acid sequence of SEQ ID NO:1. In yetanother embodiment, the nucleic acid comprises a nucleotide sequence ofSEQ ID NO:2. In yet another embodiment, the nucleic acid is from human.In yet another embodiment, the nucleic acid is amplified by primers thatselectively hybridize under stringent hybridization conditions to thesame sequence as degenerate primer sets encoding amino acid sequencesselected from the group consisting of: SEQ ID NO:3 (MLGQQQQ) and SEQ IDNO:4 (KKDRRSR). In yet another embodiment, the nucleic acid encodes apolypeptide having a molecular weight of about 28 kDa to about 38 kDa.In yet another embodiment, the nucleic acid encodes a tumor suppressorpolypeptide p33ING2 that specifically hybridizes under stringentconditions to a nucleic acid comprising a nucleotide sequence of SEQ IDNO:2. In yet another embodiment, the nucleic acid selectively hybridizesunder moderately stringent hybridization conditions to a nucleic acidcomprising a nucleotide sequence of SEQ ID NO:2.

In another aspect, the present invention provides an isolated tumorsuppressor polypeptide p33ING2, wherein the polypeptide has greater than70 % amino acid sequence identity to a polypeptide comprising an aminoacid sequence of SEQ ID NO:1. In one embodiment, the tumor suppressorpolypeptide selectively binds to polyclonal antibodies generated againsta polypeptide comprising an amino acid sequence of SEQ ID NO:1. Inanother embodiment, the polypeptide is from human. In yet anotherembodiment, the polypeptide is wildtype p33ING2.

In yet another aspect, the present invention provides an antibody thatselectively binds to a p33ING2 polypeptide comprising an amino acidsequence of SEQ ID NO:1, but does not bind to a p33ING1 polypeptidecomprising an amino acid sequence of SEQ ID NO:8. In one embodiment, theantibody is polyclonal. In another embodiment, the antibody selectivelybinds to a p33ING2 polypeptide comprising the amino acid sequence of SEQID NO:5, but does not bind to a p33ING1 polypeptide comprising an aminoacid sequence of SEQ ID NO:8.

In yet another aspect, the present invention provides an expressionvector comprising any one or more of the p33ING2 nucleic acid describedherein. The invention also provides a host cell transfected with avector comprising any one or more of the p33ING2 nucleic acid describedherein.

In yet another aspect, the present invention provides a method foridentifying a compound that modulates a tumor suppressor polypeptidep33ING2, the method comprising the steps of: (i) contacting the compoundwith a eukaryotic host cell or cell membrane in which has been expresseda tumor suppressor polypeptide p33ING2, wherein the polypeptide hasgreater than 70 % amino acid sequence identity to a polypeptidecomprising an amino acid sequence of SEQ ID NO:1; and (ii) determiningthe functional effect of the compound upon the cell or cell membraneexpressing the polypeptide. In one embodiment of the method, thepolypeptide selectively binds to polyclonal antibodies generated againsta polypeptide comprising an amino acid sequence of SEQ ID NO:1. Inanother embodiment of the method, functional effect is determined bymeasuring changes in cell growth. In yet another embodiment of themethod, the polypeptide is recombinant. In yet another embodiment of themethod, the polypeptide is from a human. In yet another embodiment ofthe method, the polypeptide comprises an amino acid sequence of SEQ IDNO:1. In yet another embodiment of the method, the cell is an HCT116human colon cancer cell line. In yet another embodiment of the method,the cell has the missense p33ING2 sequence of a polypeptide comprisingan amino acid sequence of SEQ ID NO:6.

In yet another aspect, the present invention provides a method ofinhibiting cellular proliferation, the method comprising transducing acell with an expression vector, the vector comprising a nucleic acidencoding a tumor suppressor polypeptide p33ING2, wherein the polypeptidehas greater than 70 % amino acid sequence identity to a polypeptidecomprising an amino acid sequence of SEQ ID NO:1. In one embodiment ofthe method, the polypeptide selectively binds to polyclonal antibodiesgenerated against a polypeptide comprising an amino acid sequence of SEQID NO:1. In another embodiment of the method, the nucleic acid encodes apolypeptide comprising an amino acid sequence of SEQ ID NO:1. In yetanother embodiment of the method, the nucleic acid comprises anucleotide sequence of SEQ ID NO:2. In yet another embodiment of themethod, the nucleic acid is from human. In yet another embodiment of themethod, the nucleic acid encodes a polypeptide having a molecular weightof about 28 kDa to about 38 kDa. In yet another embodiment of themethod, the cell has a missense or null endogenous p33ING2 phenotype. Inyet another embodiment of the method, the cell has a missense p33ING2sequence of a polypeptide comprising an amino acid sequence of SEQ IDNO:6.

In yet another aspect, the present invention provides a method ofdetecting the presence or absence of p33ING2 in mammalian tissue, themethod comprising the steps of: (i) isolating a biological sample; (ii)contacting the biological sample with a p33ING2-specific reagent thatselectively associates with p33ING2; and (iii) detecting the level ofp33ING2-specific reagent that selectively associates with the sample. Inone embodiment of the method, the p33ING2-specific reagent is selectedfrom the group consisting of a p33ING2-specific antibody, ap33ING2-specific primer, and a p33ING2-specific nucleic acid probe. Inanother embodiment of the method, the p33ING2-specific nucleic acidprobe binds to a nucleic acid comprising a nucleotide sequence of SEQ IDNO:7, or to a nucleic acid comprising a nucleotide sequence of SEQ IDNO:2, or to a nucleic acid comprising a nucleotide sequence of SEQ IDNO:10. In yet another embodiment of the method, the biological samplecomprises intact chromosome 4q35. In yet another embodiment of themethod, the p33ING2-specific reagent detects nucleic acid, such as DNAor RNA. In yet another embodiment of the method, the nucleic acid is apolymorphic variant of p33ING2. In yet another embodiment of the method,the p33ING2-specific reagent is an antibody that selectively binds top33ING2. In some embodiments, the antibody is polyclonal. In yet anotherembodiments of the method, the antibody selectively binds to a p33ING2polypeptide comprising an amino acid sequence of SEQ ID NO:1, but not toa p33ING1 polypeptide comprising an amino acid sequence of SEQ ID NO:8.In yet another embodiment of the method, the antibody selectively bindsto a p33ING2 polypeptide comprising an amino acid sequence of SEQ IDNO:5, but does not bind to a p33ING1 polypeptide comprising an aminoacid sequence of SEQ ID NO:8.

In yet another aspect, the present invention provides a method ofdetermining a test amount of p33ING2 in mammalian tissue, the methodcomprising the steps of: (i) isolating a biological sample; (ii)contacting the biological sample with a p33ING2-specific reagent thatselectively associates with p33ING2; and (iii) comparing the test amountto a control. In one embodiment, the control is an amount of p33ING2 ina normal cell. In another embodiment, the p33ING2-specific reagent isselected from the group consisting of p33ING2-specific antibody, ap33ING2-specific primer; and p33ING2-specific nucleic acid probe.

In yet another aspect, the present invention provides a method ofdetecting the presence or absence of p33ING1 in mammalian tissue, themethod comprising the steps of: (i) isolating a biological sample; (ii)contacting the biological sample with a p33ING1-specific antibody thatselectively binds to p33ING1 but not to p33ING2; and (iii) detecting thelevel of p33ING1-specific antibody that selectively associates with thesample. In one embodiment, the p33ING1-specific antibody is polyclonal.

In yet another aspect, the present invention provides a method ofdetermining a test amount of p33ING1 in mammalian tissue, the methodcomprising the steps of: (i) isolating a biological sample; (ii)contacting the biological sample with a p33ING1-specific antibody thatselectively associates with p33ING1 but not to p33ING2; and (iii)comparing the test amount to a control. In one embodiment, the controlis an amount of p33ING1 in a normal cell. In another embodiment, thep33ING1-specific antibody is polyclonal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates binding specificities of polyclonal antibodies forp33ING2 and polyclonal antibodies for p33ING1 by Western blot analysis.

FIG. 2 illustrates a Western blot that shows that p33ING2 protein isinduced by topoisomerase II inhibitor, etoposide.

FIG. 3 illustrates FACScan flow cytometric data that shows that p33ING1or p33ING2 can induce G₁ cell cycle arrest.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention provides for the first time nucleic acids andpolypeptides of a new tumor suppressor called p33ING2. The presentinvention also provides antibodies which selectively bind to a p33ING2protein, but not to a p33ING1 protein; and antibodies which selectivelybind to a p33ING1 protein, but not to a p33ING2 protein. These nucleicacids and the polypeptides they encode are tumor suppressors. Thesetumor suppressor nucleic acids and polypeptides are involved in theregulation of cell proliferation and in the control of cellular aging,anchorage dependence, and apoptosis.

The present invention also provides methods of screening for modulators(e.g., activators, inhibitors, stimulators, enhancers, agonists, andantagonists) of these novel p33ING2 proteins. Such modulators are usefulfor pharmacological and genetic modulation of cell growth and tumorsuppression. The invention thus provides assays for tumor suppressionand cell growth, where p33ING2 acts as a direct or indirect reportermolecule for measuring the effect of modulators on cell growth or tumorsuppression. These assays can measure various parameters that areaffected by the p33ING2 activity, e.g., cell growth on soft agar,contact inhibition and density limitation of growth, growth factor orserum dependence, tumor specific markers levels, invasiveness intoMatrigel, tumor growth in vivo, p33ING2 protein or mRNA levels,transcriptional activation or repression of a reporter gene, and thelike.

The present invention also provides methods of inhibiting cellproliferation of a cell by transducing the cell with an expressionvector containing p33ING2 nucleic acids. The transduced cell may have amissense or null endogenous p33ING2 phenotype or a mutation in anothertumor suppressor gene. For example, the cell may contain p33ING2 havinga sequence of SEQ ID NO:6 with a missense mutation. Expression ofwildtype p33ING2 restores cell growth regulation and prevents thedevelopment of tumor. For example, p33ING2 nucleic acids can be used totreat cancer or other cell proliferative diseases, such as hyperplasia,in patients.

Finally, the invention provides for methods of detecting p33ING2 orp33ING1 nucleic acid and protein expression, allowing investigation ofcell growth regulation and tumor suppression. Furthermore, p33ING2 orp33ING1 nucleic acid and protein expression can be used to diagnosecancer in patients who have a defect in one or more copies of p33ING2 orp33ING1 in their genome.

Functionally, p33ING2 represents a nuclear protein having a molecularweight of approximately 33 kDa. It is involved in the regulation of cellproliferation and in the control of cellular aging, anchorage andapoptosis.

Structurally, the nucleotide sequence of p33ING2 (see, e.g., SEQ IDNO:2, isolated from a human) encodes a polypeptide of approximately 270amino acids with a predicted molecular weight of approximately 33 kDaand a predicted range of 28-38 kDa (see, e.g., SEQ ID NO:1). Relatedp33ING2 genes from other species share at least about 70% amino acididentity over an amino acid region of at least about 25 amino acids inlength, preferably 50 to 100 amino acids in length.

Specific regions of the p33ING2 nucleotide and amino acid sequences maybe used to identify polymorphic variants, interspecies homologs, andalleles of p33ING2. This identification can be made in vitro, e.g.,under stringent hybridization conditions or with PCR and sequencing, orby using the sequence information in a computer system for comparisonwith other nucleotide or amino acid sequences. Typically, identificationof polymorphic variants and alleles of p33ING2 is made by comparing anamino acid sequence of about 25 amino acids or more, preferably 50-100amino acids. Amino acid identity of approximately at least 70% or above,preferably 80%, most preferably 90-95% or above typically demonstratesthat a protein is a polymorphic variant, interspecies homolog, or alleleof p33ING2. Sequence comparison can be performed using any of thesequence comparison algorithms discussed below. Antibodies that bindspecifically to p33ING2 or a conserved region thereof can also be usedto identify alleles, interspecies homologs, and polymorphic variants.

Polymorphic variants, interspecies homologs, and alleles of p33ING2 areconfirmed by examining the effect of putative p33ING2 expression on cellgrowth and tumor suppression using the methods and assays describedherein. Typically, p33ING2 having the amino acid sequence of SEQ ID NO:1is used as a positive control. For example, immunoassays usingantibodies directed against the amino acid sequence of SEQ ID NOS:1 or 5can be used to demonstrate the identification of a polymorphic variantor allele of p33ING2. Alternatively, p33ING2 having the nucleic acidsequences of SEQ ID NO:1 is used as a positive control, e.g., in in situhybridization with SEQ ID NO:1 to demonstrate the identification of apolymorphic variant or allele of p33ING2. The polymorphic variants,alleles and interspecies homologs of p33ING2 are expected to retain theability to inhibit cell proliferation and tumor suppression. Thesefunctional characteristics can be tested using various assays, such assoft agar assay, contact inhibition and density limitation of growthassay, growth factor or serum dependence assay, tumor specific markersassay, invasiveness assay, apoptosis assay, G₀/G₁ cell cycle arrestassay, tumor growth assay, etc.

The present invention also provides polymorphic variants of p33ING2depicted in SEQ ID NO:1: variant #1, in which a threonine residue issubstituted for a serine residue at amino acid position 11; variant #2,in which a leucine residue is substituted for an isoleucine residue atamino acid position 101; and variant #3, in which an alanine residue issubstituted for a glycine residue at amino acid position 251.

P33ING2 nucleotide and amino acid sequence information may also be usedto construct models of tumor suppressor polypeptides in a computersystem. These models are subsequently used to identify compounds thatcan activate or inhibit p33ING2. Such compounds that modulate theactivity of p33ING2 can be used to investigate the role of p33ING2 ininhibition of cell proliferation and tumor suppression or can be used astherapeutics.

Isolation of p33ING2 provides a means for assaying for modulators ofp33ING2. P33ING2 is useful for testing modulators using in vivo and invitro expression that measure various parameters, e.g., cell growth onsoft agar, contact inhibition and density limitation of growth, growthfactor or serum dependence, tumor specific markers levels, invasivenessinto Matrigel, apoptosis assay, G₀/G₁ cell cycle arrest assay, tumorgrowth in vivo, p33ING2 protein or mRNA levels, transcriptionalactivation or repression of a reporter gene, and the like. Suchmodulators identified using p33ING2 can be used to study cell growthregulation and tumor suppression, and further to treat cancer.

Methods of detecting p33ING2 nucleic acids and expression of p33ING2 arealso useful for diagnosing various cancers or tumors by using assayssuch as northern blotting, dot blotting, in situ hybridization, RNaseprotection, and the like. Chromosome localization of the genes encodinghuman p33ING2 can also be used to identify diseases, mutations, andtraits caused by and associated with p33ING2. Techniques, such as highdensity oligonucleotide arrays (GeneChip™), can be also be used toscreen for mutations, polymorphic variants, alleles and interspecieshomologs of p33ING2.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The term “tumor suppressor” refers to a gene, or the protein it encodes,that in its wildtype form has the ability to suppress, prevent, ordecrease cell transformation. Tumor suppressor genes are genes thatencode protein(s) that regulate cell growth and proliferation directlyor indirectly, e.g., p53, Rb, and the like. If a tumor suppressor geneis damaged (e.g., by radiation , a carcinogen or inherited, orspontaneous mutation), it may lose its wildtype ability to regulate cellgrowth and proliferation, and the cells may become transformed orpre-disposed to transformation.

“p33ING” refers to a family of tumor suppressor nucleic acids orpolypeptides having a molecular weight of approximately 33 kDa. Theyencode a nuclear protein which is involved in the regulation of cellgrowth and proliferation and in the control of cellular aging, anchorageand apoptosis. “p33ING2” and “p33ING1” are members of the “p33ING”family, which members are encoded by different genes (i.e., mapped todifferent regions on the chromosome). p33ING2 is mapped to humanchromosome 4q35.

The term p33ING2 therefore refers to polymorphic variants, alleles,interspecies homologs, and mutants that: (1) have about 70% amino acidsequence identity, preferably about 80-90% amino acid sequence identityto SEQ ID NO:1 over a window of about at least 50-100 amino acids; (2)binds to polyclonal antibodies raised against an immunogen comprising anamino acid sequence selected from the group consisting of SEQ ID NO:1and conservatively modified variants thereof, but does not bind topolyclonal antibodies raised against an immunogen comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:8 andconservatively modified variants thereof; (3) specifically hybridize(with a size of at least about 500, preferably at least about 900nucleotides) under stringent hybridization conditions to a sequenceselected from the group consisting of SEQ ID NO:2, and conservativelymodified variants thereof; or (4) are amplified by primers thatspecifically hybridize under stringent conditions to the same sequenceas a degenerate primers sets encoding SEQ ID NOS:3 and 4.

The term p33ING1 refers to polymorphic variants, alleles, interspecieshomologs, and mutants that: (1) have about 70% amino acid sequenceidentity, preferably about 80-90% amino acid sequence identity to SEQ IDNO:8 over a window of about at least 50-100 amino acids; (2) binds topolyclonal antibodies raised against an immunogen comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:8 andconservatively modified variants thereof, but does not bind topolyclonal antibodies raised against an immunogen comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:1 andconservatively modified variants thereof.

The phrases “polymorphic variant” and “allele” refer to forms of p33ING2that occur in a population (or among populations) and that maintainwildtype p33ING2 activity as measured using one of the assays describedherein.

The term “mutant” of p33ING2 refers to those mutants which areexperimentally made or those which are found in tumor or cancer cells.Mutants of p33ING2 can be due to, e.g., truncation, elongation,substitution of amino acids, deletion, insertion, or lack of expression(e.g., due to promoter or splice site mutations, etc.). A mutant hasactivity that differs from the activity of wildtype p33ING2 by at leastabout 20% as measured using an assay described herein. For example, amutant of p33ING2 can have a null mutation which results in absence ofnormal gene product at the molecular level or an absence of function atthe phenotypic level. Another example is a missense mutation of p33ING2,where a substitution of amino acid(s) results in a change in theactivity of the protein.

The phrase “missense or null endogenous p33ING2 phenotype” of a celltherefore refers to p33ING2 has a missense or null mutation so that thecell has a phenotype (e.g., soft agar growth, contact inhibition anddensity limitation of growth, etc.) which differs from a cell having awildtype p33ING2.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The term “transfect” or “transduce” refers to any way of getting anucleic acid across a cell membrane, including electroporation,biolistics, injection, plasmid transfection, lipofection, viraltransduction, lipid-nucleic acid complexes, naked DNA, etc

A “host cell” is a naturally occurring cell or a transformed cell thatcontains an expression vector and supports the replication or expressionof the expression vector. Host cells may be cultured cells, explants,cells in vivo, and the like. Host cells may be prokaryotic cells such asE. Coli, or eukaryotic cells such as yeast, insect, amphibian, ormammalian cells such as CHO, HeLa, HCT116, RK0 cells, and the like.

“Biological sample” include, but are not limited to, tissue isolatedfrom humans, mice, and rats. In some embodiments, a sample of biologicaltissue or fluid contains nucleic acids or polypeptides of p33ING2 and/orp33ING1. Biological samples may also include sections of tissues such asfrozen sections taken from histological purposes. A biological sample istypically obtained from a eukaryotic organism, such as insects,protozoa, birds, fish, reptiles, and preferably a mammal such as rat,mouse, cow, dog, guinea pig, or rabbit, and most preferably a primatesuch as chimpanzees or humans.

“Tumor cell” refers to precancerous, cancerous, and normal cells in atumor.

“Cancer cells”, “transformed” cells or “transformation” in tissueculture, refers to spontaneous or induced phenotypic changes that do notnecessarily involve the uptake of new genetic material. Althoughtransformation can arise from infection with a transforming virus andincorporation of new genomic DNA, or uptake of exogenous DNA, it canalso arise spontaneously or following exposure to a carcinogen, therebymutating an endogenous gene. Transformation is associated withphenotypic changes, such as immortalization of cells, aberrant growthcontrol, and/or malignancy (see, Freshney, Culture of Animal Cells aManual of basic Technique (3^(rd) ed. 1994)).

The term “cell cycle” refers to the cyclic biochemical and structuralevens occurring during growth of cells. The cell cycle is divided intoperiods called: G₀, Gap₁ (G₁), DNA synthesis (S), GAP₂ (G₂), and mitosis(M).

The phrase “functional effects” in the context of assays for testingcompounds that modulate p33ING2 mediated tumor suppression includes thedetermination of any parameter that is indirectly or directly under theinfluence of the p33ING2 protein. Functional effects include, e.g.,anchorage dependence, contact inhibition and density limitation ofgrowth, growth factor or serum dependence, tumor specific markerslevels, invasiveness, tumor growth, p33ING2 protein mRNA levels,apoptosis, G₀/G₁ cell cycle arrest, and the like, in vitro, in vivo, andex vivo.

By “determining the functional effect” is meant assays for a compoundthat increases or decreases a parameter that is directly or indirectlyunder the influence of p33ING2. Such functional effects can be measuredby any means known to those skilled in the art, e.g., soft agar assay,contact inhibition and density limitation of growth assay, growth factoror serum dependence assay, tumor specific markers assay, invasivenessassay, apoptosis assay, G₀/G₁ cell cycle arrest assay, tumor growthassay, p33ING2 protein mRNA level assay, transcriptional activation orrepression of a reporter gene assay, and the like, in vitro, in vivo,and ex vivo.

“Inhibitors,” “activators,” and “modulators” of p33ING2 refer toinhibitory, activating, or modulatory molecules identified using invitro and in vivo assays for tumor suppression, e.g., ligands, agonists,antagonists, and their homologs and mimetics. Inhibitors are compoundsthat decrease, block, prevent, delay activation, inactivate,desensitize, or down regulate tumor suppression, e.g., antagonists.Activators are compounds that increase, activate, facilitate, enhanceactivation, sensitize or up regulate tumor suppression, e.g., agonists.Modulators are inhibitors and activators and include geneticallymodified versions of p33ING2, e.g., with altered activity, as well asnaturally occurring and synthetic ligands, antagonists, agonists, smallchemical molecules and the like. Such assays for modulators include,e.g., expressing p33ING2 in cells, applying putative modulatorcompounds, and then determining the functional effects on inhibition ofcell proliferation or tumor suppression. Compounds identified by theseassays are used to modulate tumor suppression effect of p33ING2.

Samples or assays comprising p33ING2 that are treated with a potentialmodulator are compared to control samples without the inhibitor,activator, or modulator. Control samples (untreated with inhibitors) areassigned a relative p33ING2 activity value of 100%. Inhibition ofp33ING2 is achieved when the p33ING2 activity value relative to thecontrol is about 90% or less, optionally about 80% or less, 70% or less,60% or less, 50% or less, 40% or less, 30% or less, or 25-0%. Activationof p33ING2 is achieved when the p33ING2 activity value relative to thecontrol (untreated with activators) is 110% or more, optionally 120%,130%, 140%, 150% or more, 200-500% or more, 1000-3000% or more.

The phrase “changes in cell growth” refers to any change in cell growthand proliferation characteristics in vitro or in vivo, such as formationof foci, anchorage independence, semi-solid or soft agar growth, changesin contact inhibition and density limitation of growth, loss of growthfactor or serum requirements, changes in cell morphology, gaining orlosing immortalization, gaining or losing tumor specific markers,ability to form or suppress tumors when injected into suitable animalhosts, and/or immortalization of the cell. See, e.g., Freshney, Cultureof Animal Cells a Manual of Basic Technique, 3^(rd) ed. (Wiley-Liss,Inc. 1994), pp.231-241, herein incorporated by reference. The phrase“changes in cell growth” can also refer to changes in apoptosis orchanges in cell cycle pattern.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription.

A “constitutive” promoter is a promoter that is active under mostenvironmental and developmental conditions. An “inducible” promoter is apromoter that is active under environmental or developmental regulation.

The term “operably linked” refers to a functional linkage between anucleic acid expression control sequence (such as a promoter, or arrayof transcription factor binding sites) and a second nucleic acidsequence, wherein the expression control sequence directs transcriptionof the nucleic acid corresponding to the second sequence.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

A “label” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include 32P, fluorescent dyes, electron-dense reagents, enzymes(e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptensand proteins for which antisera or monoclonal antibodies are available(e.g., the polypeptide of SEQ ID NO:1 can be made detectable, e.g., byincorporating a radiolabel into the peptide, and used to detectantibodies specifically reactive with the peptide).

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components whichnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified. Inparticular, an isolated p33ING2 nucleic acid is separated from openreading frames that flank the p33ING2 gene and encode proteins otherthan p33ING2. The term “purified” denotes that a nucleic acid or proteingives rise to essentially one band in an electrophoretic gel.Particularly, it means that the nucleic acid or protein is at least 85%pure, more preferably at least 95% pure, and most preferably at least99% pure.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. The term nucleic acid is usedinterchangeably with gene, cDNA, mRNA, oligonucleotide, andpolynucleotide.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an analog or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group. Examples ofamino acid analogs include homoserine, norleucine, methionine sulfoxide,methionine methyl sulfonium. Such analogs have modified R groups (e.g.,norleucine) or modified peptide backbones, but retain the same basicchemical structure as a naturally occurring amino acid. Amino acidmimetics refers to chemical compounds that have a structure that isdifferent from the general chemical structure of an amino acid, but thatfunction in a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refer to those nucleic acidswhich encode identical or essentially identical amino acid sequences.Where the nucleic does not encode an amino acid sequence (e.g., aribosomal RNA), conservatively modified variants refer to essentiallyidentical sequences. Specifically, degenerate codon substitutions may beachieved by generating sequences in which the third position of one ormore selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al.,Mol. Cell. Probes 8:91-98 (1994)). Because of the degeneracy of thegenetic code, a large number of functionally identical nucleic acidsencode any given protein. For instance, the codons GCA, GCC, GCG and GCUall encode the amino acid alanine. Thus, at every position where analanine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Glycine (G);

2) Serine (S), Threonine (T);

3) Aspartic acid (D), Glutamic acid (E);

4) Asparagine (N), Glutamine (Q);

5) Cysteine (C), Methionine (M);

6) Arginine (R), Lysine (K), Histidine (H);

7) Isoleucine (I), Leucine (L), Valine (V); and

8) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (see, e.g.,Creighton, Proteins (1984)).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 70% identity, preferably 75%, 80%, 85%, 90%, or 95% identity orhigher over a specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to thecompliment of a test sequence. Preferably, the identity exists over aregion that is at least about 25 amino acids or nucleotides in length,or more preferably over a region that is 50-100 amino acids ornucleotides in length. In most preferred embodiments, the sequences aresubstantially identical over the entire length of, e.g., the codingregion.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

Another example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a tree or dendogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987). The method used is similar to themethod described by Higgins & Sharp, CABIOS 5:151-153 (1989). Theprogram can align up to 300 sequences, each of a maximum length of 5,000nucleotides or amino acids. The multiple alignment procedure begins withthe pairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-395 (1984)).

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal is at least two times background, preferably 10 timesbackground hybridization. Exemplary stringent hybridization conditionscan be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42°C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and0.1% SDS at 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, I, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H)l by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab)′₂ dimer intoan Fab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993)). Whilevarious antibody fragments are defined in terms of the digestion of anintact antibody, one of skill will appreciate that such fragments may besynthesized de novo either chemically or by using recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Coleet al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc. (1985)). Techniques for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produceantibodies to polypeptides of this invention. Also, transgenic mice, orother organisms such as other mammals, may be used to express humanizedantibodies. Alternatively, phage display technology can be used toidentify antibodies and heteromeric Fab fragments that specifically bindto selected antigens (see, e.g., McCafferty et al., Nature 348, 552-554(1990); Marks et al., Biotechnology 10, 779-783 (1992)).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

An “anti-p33ING2” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by the p33ING2 gene, cDNA, or asubsequence thereof.

An “anti-p33ING1” antibody is an antibody or antibody fragment thatspecifically binds to a polypeptide encoded by the p33ING1 gene, cDNA,or a subsequence thereof.

The term “immunoassay” is an assay that uses an antibody to specificallybind an antigen. The immunoassay is characterized by the use of specificbinding properties of a particular antibody to isolate, target, and/orquantify the antigen.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to p33ING2 at least two times the background,more typically 10 to 100 times background, and do not substantially bindin a significant amount to other proteins present in the sample.Specific binding to a polyclonal antibody under such conditions mayrequire an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies raised to p33ING2from a species such as rat, mouse, or human can be selected to obtainonly those polyclonal antibodies that are specifically immunoreactivewith p33ING2 and not with other proteins, such as p33ING1, except forpolymorphic variants and alleles of p33ING2. This selection may beachieved for polyclonal antibodies by subtracting out antibodies thatcross react with p33ING1. For monoclonal antibodies, the specificity maybe achieved by using a p33ING2 specific antigen to make the hybridomas(e.g., SEQ ID NO:5). See, e.g., FIG. 2. Similarly, polyclonal antibodiesraised to p33ING1 from a species such as rat, mouse, or human can beselected to obtain only those polyclonal antibodies that arespecifically immunoreactive with p33ING1 and not with other proteins,such as p33ING2, except for polymorphic variants and alleles of p33ING1using the methods described above. For identifying p33ING2 or p33ING1variants and alleles from a particular species such as a human, theselection may be achieved by subtracting out antibodies that cross-reactwith p33ING2 or p33ING1 molecules, respectively, from other species. Forspecies specific monoclonal antibodies, a species specific antigen canbe used to make the hybridomas. A variety of immunoassay formats may beused to select antibodies specifically immunoreactive with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select antibodies specifically immunoreactive with a protein (see,e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity).

The phrase “selectively associates with” refers to the ability of anucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

“p33ING2-specific reagent” refers to any reagent which specificallyassociates with p33ING2. For example, it can be a p33ING2-specificantibody, a p33ING2-specific primer, or a p33ING2-specific nucleic acidprobe.

III. Isolation of the Gene Encoding p33ING2

A. General recombinant DNA methods

P33ING2 polypeptides and nucleic acids are used in the assays describedbelow. For example, recombinant p33ING2 can be used to make cells thatconstitutively express p33ING2. Such polypeptides and nucleic acids canbe made using routine techniques in the field of recombinant genetics.Basic texts disclosing the general methods of use in this inventioninclude Sambrook et al., Molecular Cloning, A Laboratory Manual (2^(nd)ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual(1990); and Current Protocols in Molecular Biology (Ausubel et al.,eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides can be chemically synthesized according to the solidphase phosphoramidite triester method first described by Beaucage &Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automatedsynthesizer, as described in Van Devanter et al., Nucleic Acids Res.12:6159-6168 (1984). Purification of oligonucleotides is typically byeither native acrylamide gel electrophoresis or by anion-exchange HPLCas described in Pearson & Reanier, J. Chrom. 255:137-149 (1983). Thesequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981). Again, as noted above, companies such as Operon Technologies,Inc. provide an inexpensive commercial source for essentially anyoligonucleotide.

B. Cloning Methods for the isolation of nucleotide sequences encodingp33ING2

In general, the nucleic acid sequences encoding genes of interest, suchas p33ING2 and related nucleic acid sequence homologs, are cloned fromcDNA and genomic DNA libraries by hybridization with a probe, orisolated using amplification techniques with oligonucleotide primers.Preferably mammalian, more preferably human sequences are used. Forexample, p33ING2 sequences are typically isolated from mammalian nucleicacid (genomic or cDNA) libraries by hybridizing with a nucleic acidprobe, the sequence of which can be derived from SEQ ID NO:1. A suitabletissue from which human p33ING2 RNA and cDNA can be isolated is, e.g.,placenta, HepG2 or Saos-2 cell lines.

Amplification techniques using primers can also be used to amplify andisolate, e.g., a nucleic acid encoding p33ING2, from DNA or RNA (see,e.g., Dieffenfach & Dveksler, PCR Primer: A Laboratory Manual (1995)).These primers can be used, e.g., to amplify either the full lengthsequence or a probe of one to several hundred nucleotides, which is thenused to screen a mammalian library for the full-length nucleic acid ofchoice. For example, degenerate primer sets, such as MLGQQQQ (SEQ IDNO:3) and KKDRRSR (SEQ ID NO:4) can be used to isolate p33ING2 nucleicacids. Nucleic acids can also be isolated from expression librariesusing antibodies as probes. Such polyclonal or monoclonal antibodies canbe raised, e.g., using the sequence of p33ING2.

Polymorphic variants and alleles that are substantially identical to thegene of choice can be isolated using nucleic acid probes, andoligonucleotides under stringent hybridization conditions, by screeninglibraries. Alternatively, expression libraries can be used to clone,e.g., p33ING2 and p33ING2 polymorphic variants, interspecies homologs,and alleles, by detecting expressed homologs immunologically withantisera or purified antibodies made against p33ING2, which alsorecognize and selectively bind to the p33ING2 homolog.

To make a cDNA library, one should choose a source that is rich in themRNA of choice, e.g., for human p33ING2 mRNA, placenta, HepG2 or Saos-2cell lines. The mRNA is then made into cDNA using reverse transcriptase,ligated into a recombinant vector, and transfected into a recombinanthost for propagation, screening and cloning. Methods for making andscreening cDNA libraries are well known (see, e.g., Gubler & Hoffman,Gene 25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra).

For a genomic library, the DNA is extracted from the tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in non-lambdaexpression vectors. These vectors are packaged in vitro. Recombinantphage are analyzed by plaque hybridization as described in Benton &Davis, Science 196:180-182 (1977). Colony hybridization is carried outas generally described in Grunstein et al., Proc. Natl. Acad. Sci. USA.,72:3961-3965 (1975).

An alternative method of isolating a nucleic acid and its homologscombines the use of synthetic oligonucleotide primers and amplificationof an RNA or DNA template (see U.S. Pat. Nos. 4,683,195 and 4,683,202;PCR Protocols: A Guide to Methods and Applications (Innis et al., eds,1990)). Methods such as polymerase chain reaction (PCR) and ligase chainreaction (LCR) can be used to amplify nucleic acid sequences of, e.g.,p33ING2 directly from mRNA, from cDNA, from genomic libraries or cDNAlibraries. Degenerate oligonucleotides can be designed to amplifyp33ING2 homologs using the sequences provided herein. Restrictionendonuclease sites can be incorporated into the primers. Polymerasechain reaction or other in vitro amplification methods may also beuseful, for example, to clone nucleic acid sequences that code forproteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of p33ING2 encoding mRNA in physiologicalsamples, for nucleic acid sequencing, or for other purposes. Genesamplified by the PCR reaction can be purified from agarose gels andcloned into an appropriate vector.

As described above, gene expression of p33ING2 or p33ING1 can also beanalyzed by techniques known in the art, e.g., reverse transcription andPCR amplification of mRNA, isolation of total RNA or poly A+ RNA,northern blotting, dot blotting, in situ hybridization, RNaseprotection, probing high density oligonucleotides, and the like. All ofthese techniques are standard in the art.

Synthetic oligonucleotides can be used to construct recombinant genesfor use as probes or for expression of protein. This method is performedusing a series of overlapping oligonucleotides usually 40-120 bp inlength, representing both the sense and non-sense strands of the gene.These DNA fragments are then annealed, ligated and cloned.Alternatively, amplification techniques can be used with precise primersto amplify a specific subsequence of the p33ING2 nucleic acid. Thespecific subsequence is then ligated into an expression vector.

The nucleic acid encoding the protein of choice is typically cloned intointermediate vectors before transformation into prokaryotic oreukaryotic cells for replication and/or expression. These intermediatevectors are typically prokaryote vectors, e.g., plasmids, or shuttlevectors. Optionally, cells can be transfected with recombinant p33ING2operably linked to a constitutive promoter, to provide higher levels ofp33ING2 expression in cultured cells.

C. Expression in prokaryotes and eukaryotes

To obtain high level expression of a cloned gene or nucleic acid, suchas those cDNAs encoding p33ING2, one typically subclones p33ING2 into anexpression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. and Ausubel et al.Bacterial expression systems for expressing the p33ING2 protein areavailable in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al.,Gene 22:229-235 (1983)). Kits for such expression systems arecommercially available. Eukaryotic expression systems for mammaliancells, yeast, and insect cells are well known in the art and are alsocommercially available.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is preferablypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function. Thepromoter typically cam also include elements that are responsive totransactivation, e.g., hypoxia responsive elements, Gal4 responsiveelements, lac repressor responsive elements, and the like. The promotercan be constitutive or inducible, heterologous or homologous.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the nucleic acid inhost cells. A typical expression cassette thus contains a promoteroperably linked, e.g., to.the nucleic acid sequence encoding p33ING2,and signals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. The nucleic acidsequence may typically be linked to a cleavable signal peptide sequenceto promote secretion of the encoded protein by the transformed cell.Such signal peptides would include, among others, the signal peptidesfrom tissue plasminogen activator, insulin, and neuron growth factor,and juvenile hormone esterase of Heliothis virescens. Additionalelements of the cassette may include enhancers and, if genomic DNA isused as the structural gene, introns with functional splice donor andacceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical (one expressionvector is described in Example I). Any of the conventional vectors usedfor expression in eukaryotic or prokaryotic cells may be used. Standardbacterial expression vectors include plasmids such as pBR322 basedplasmids, pSKF, pET23D, and fusion expression systems such as GST andLacZ. Epitope tags can also be added to recombinant proteins to provideconvenient methods of isolation, e.g., c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+,pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable, such as using abaculovirus vector in insect cells, with a p33ING2 encoding sequenceunder the direction of the polyhedrin promoter or other strongbaculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of protein,which are then purified using standard techniques (see, e.g., Colley etal., J. Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Any of the well known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (see,e.g., Sambrook et al., supra). It is only necessary that the particulargenetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingthe protein of choice.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofthe p33ING2 protein, which is recovered from the culture using standardtechniques identified below.

IV. Purification of p33ING2

If necessary, naturally occurring or recombinant proteins can bepurified for use in functional assays, e.g., to make antibodies todetect p33ING2. Naturally occurring p33ING2 is purified, e.g., frommammalian tissue such as placenta, HepG2 or Saos-2 cell lines or anyother source of a p33ING2 bomolog. Recombinant p33ING2 is purified fromany suitable expression system, e.g., by expressing p33ING2 in E. coliand then purifying the recombinant protein via affinity purification,e.g., by using antibodies that recognize a specific epitope on theprotein or on part of the fusion protein, or by using glutathioneaffinity gel, which binds to GST. In some embodiments, the recombinantprotein is a fusion protein, e.g., with GST or Gal4 at the N-terminus.

The protein of choice may be purified to substantial purity by standardtechniques, including selective precipitation with such substances asammonium sulfate; column chromatography, immunopurification methods, andothers (see, e.g., Scopes, Protein Purification: Principles and Practice(1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook etal., supra).

A number of procedures can be employed when recombinant protein is beingpurified. For example, proteins having established molecular adhesionproperties can be reversibly fused to p33ING2. With the appropriateligand, p33ING2 can be selectively adsorbed to a purification column andthen freed from the column in a relatively pure form. The fused proteinis then removed by enzymatic activity. Finally, p33ING2 could bepurified using immunoaffinity columns.

A. Purification of p33ING2 from recombinant bacteria

Recombinant proteins are expressed by transformed bacteria in largeamounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is one example of aninducible promoter system. Bacteria are grown according to standardprocedures in the art. Fresh or frozen bacteria cells are used forisolation of protein.

Proteins expressed in bacteria may form insoluble aggregates (“inclusionbodies”). Several protocols are suitable for purification of inclusionbodies. For example, purification of inclusion bodies typically involvesthe extraction, separation and/or purification of inclusion bodies bydisruption of bacterial cells, e.g., by incubation in a buffer of 50 mMTRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 0.1 mM ATP, and 1 mMPMSF. The cell suspension can be lysed using 2-3 passages through aFrench press, homogenized using a Polytron (Brinkman Instruments) orsonicated on ice. Alternate methods of lysing bacteria are apparent tothose of skill in the art (see, e.g., Sambrook et al., supra; Ausubel etal., supra).

If necessary, the inclusion bodies are solubilized, and the lysed cellsuspension is typically centrifuged to remove unwanted insoluble matter.Proteins that formed the inclusion bodies may be renatured by dilutionor dialysis with a compatible buffer. Suitable solvents include, but arenot limited to urea (from about 4 M to about 8 M), formamide (at leastabout 80%, volume/volume basis), and guanidine hydrochloride (from about4 M to about 8 M). Some solvents which are capable of solubilizingaggregate-forming proteins, for example SDS (sodium dodecyl sulfate),70% formic acid, are inappropriate for use in this procedure due to thepossibility of irreversible denaturation of the proteins, accompanied bya lack of immunogenicity and/or activity. Although guanidinehydrochloride and similar agents are denaturants, this denaturation isnot irreversible and renaturation may occur upon removal (by dialysis,for example) or dilution of the denaturant, allowing re-formation ofimmunologically and/or biologically active protein. Other suitablebuffers are known to those skilled in the art. The protein of choice isseparated from other bacterial proteins by standard separationtechniques, e.g., with Ni-NTA agarose resin.

Alternatively, it is possible to purify the recombinant p33ING2 proteinfrom bacteria periplasm. After lysis of the bacteria, when the proteinis exported into the periplasm of the bacteria, the periplasmic fractionof the bacteria can be isolated by cold osmotic shock in addition toother methods known to skill in the art. To isolate recombinant proteinsfrom the periplasm, the bacterial cells are centrifuged to form apellet. The pellet is resuspended in a buffer containing 20% sucrose. Tolyse the cells, the bacteria are centrifuged and the pellet isresuspended in ice-cold 5 mM MgSO₄ and kept in an ice bath forapproximately 10 minutes. The cell suspension is centrifuged and thesupernatant decanted and saved. The recombinant proteins present in thesupernatant can be separated from the host proteins by standardseparation techniques well known to those of skill in the art.

B. Standard protein separation techniques for purifying p33ING2

Solubility fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size differential filtration

The molecular weight of the protein, e.g., p33ING2, can be used toisolated the protein from proteins of greater and lesser size usingultrafiltration through membranes of different pore size (for example,Amicon or Millipore membranes). As a first step, the protein mixture isultrafiltered through a membrane with a pore size that has a lowermolecular weight cut-off than the molecular weight of the protein ofinterest. The retentate of the ultrafiltration is then ultrafilteredagainst a membrane with a molecular cut off greater than the molecularweight of the protein of interest. The recombinant protein will passthrough the membrane into the filtrate. The filtrate can then bechromatographed as described below.

Column chromatography

The protein of choice can also be separated from other proteins on thebasis of its size, net surface charge, hydrophobicity, and affinity forligands. In addition, antibodies raised against proteins can beconjugated to column matrices and the proteins immunopurified. All ofthese methods are well known in the art. It will be apparent to one ofskill that chromatographic techniques can be performed at any scale andusing equipment from many different manufacturers (e.g., PharmaciaBiotech).

V. Immunological Detection of p33ING2 and p33ING1

In addition to the detection of p33ING2 genes and gene expression usingnucleic acid hybridization technology, one can also use immunoassays todetect p33ING2, e.g., to identify alleles, mutants, polymorphic variantsand interspecies homologs of p33ING2. Immunoassays can be used toqualitatively or quantitatively analyze p33ING2, e.g., to detectp33ING2, to measure p33ING2 activity, or to identify modulators ofp33ING2 activity. Similarly, immunoassay can be used to detect andanalyze p33ING1. A general overview of the applicable technology can befound in Harlow and Lane, Antibodies: A Laboratory Manual (1988).

A. Antibodies to p33ING2 and p33ING1

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with p33ING2 or p33ING1 are known to those of skill in theart (see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow& Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice(2^(nd) ed. 1986); and Kohler & Milstein, Nature 256:495-497 (1975)).Such techniques include antibody preparation by selection of antibodiesfrom libraries of recombinant antibodies in phage or similar vectors, aswell as preparation of polyclonal and monoclonal antibodies byimmunizing rabbits or mice (see, e.g., Huse et al., Science246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989)). Inaddition, as noted above, many companies, such as BMA Biomedicals, Ltd.,HTI Bio-products, and the like, provide the commercial service of makingan antibody to essentially any peptide.

A number of p33ING2 or p33ING1 comprising immunogens may be used toproduce antibodies specifically reactive with p33ING2 or p33ING1,respectively. For example; recombinant p33ING2 or p33ING1, or antigenicfragments thereof, are isolated as described herein. Recombinant proteincan be expressed in eukaryotic or prokaryotic cells as described above,and purified as generally described above. Recombinant protein is thepreferred immunogen for the production of monoclonal or polyclonalantibodies. Alternatively, a synthetic peptide derived from thesequences disclosed herein and conjugated to a carrier protein can beused an immunogen. Naturally occurring protein may also be used eitherin pure or impure form. The product is then injected into an animalcapable of producing antibodies. Either monoclonal or polyclonalantibodies may be generated, for subsequent use in immunoassays tomeasure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. To improve reproducibility, an inbred strain of mice(e.g., BALB/C mice) can be immunized to make the antibody; however,standard animals (mice, rabbits, etc.) used to make antibodies areimmunized with the protein using a standard adjuvant, such as Freund'sadjuvant, and a standard immunization protocol (see Harlow & Lane,supra). The animal's immune response to the immunogen preparation ismonitored by taking test bleeds and determining the titer of reactivityto the protein of choice. When appropriately high titers of antibody tothe immunogen are obtained, blood is collected from the animal andantisera are prepared. Further fractionation of the antisera to enrichfor antibodies reactive to the protein can be done if desired (seeHarlow & Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see Kohler & Milstein, Eur. J. Immunol. 6:511-519 (1976)).Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods wellknown in the art. Colonies arising from single immortalized cells arescreened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse et al.,Science 246:1275-1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-p33ING2proteins or even other related proteins, e.g., from other organisms,using a competitive binding immunoassay. Specific polyclonal antiseraand monoclonal antibodies will usually bind with K_(D) of at least about0.1 mM, more usually at least about 1 μM, preferably at least about 0.1μM or better, and most preferably, 0.01 μM or better.

Once p33ING2 or p33ING1 specific antibodies are available, theseproteins can be detected by a variety of immunoassay methods. For areview of immunological and immunoassay procedures, see Basic andClinical Immunology (Stites & Terr eds., 7^(th) ed. 1991). Moreover, theimmunoassays of the present invention can be performed in any of severalconfigurations, which are reviewed extensively in Enzyme Immunoassay(Maggio, ed., 1980); and Harlow & Lane, supra.

B. Immunological binding assays

P33ING2 or p33ING1 can be detected and/or quantified using any of anumber of well recognized immunological binding assays (see, e.g., U.S.Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a reviewof the general immunoassays, see also Methods in Cell Biology:Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic andClinical Immunology (Stites & Terr, eds., 7th ed. 1991). Immunologicalbinding assays (or immunoassays) typically use an antibody thatspecifically binds to a protein or antigen of choice (in this casep33ING2, p33ING1, or antigenic fragments thereof). The antibody may beproduced by any of a number of means well known to those of skill in theart and as described above.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled p33ING2 or p33ING1polypeptide or a labeled anti-p33ING2 or anti-p33ING1 antibody.Alternatively, the labeling agent may be a third moiety, such asecondary antibody, that specifically binds to the antibody/antigencomplex (a secondary antibody is typically specific to antibodies of thespecies from which the first antibody is derived). Other proteinscapable of specifically binding immunoglobulin constant regions, such asprotein A or protein G may also be used as the label agent. Theseproteins exhibit a strong non-immunogenic reactivity with immunoglobulinconstant regions from a variety of species (see, e.g., Kronval et al.,J. Immunol. 111:1401-1406 (1973); Akerstrom et al., J. Immunol.135:2589-2542 (1985)). The labeling agent can be modified with adetectable moiety, such as biotin, to which another molecule canspecifically bind, such as streptavidin. A variety of detectablemoieties are well known to those skilled in the art.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antigen, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

Non-competitive assay formats

Immunoassays for detecting p33ING2 or p33ING1 in samples may be eithercompetitive or noncompetitive. Noncompetitive immunoassays are assays inwhich the amount of antigen is directly measured. In one preferred“sandwich” assay, for example, the anti-antigen antibodies can be bounddirectly to a solid substrate on which they are immobilized. Theseimmobilized antibodies then capture antigen present in the test sample.Antigen thus immobilized is then bound by a labeling agent, such as asecond antibody bearing a label. Alternatively, the second antibody maylack a label, but it may, in turn, be bound by a labeled third antibodyspecific to antibodies of the species from which the second antibody isderived. The second or third antibody is typically modified with adetectable moiety, such as biotin, to which another moleculespecifically binds, e.g., streptavidin, to provide a detectable moiety.

Competitive assay formats

In competitive assays, the amount of p33ING2 or p33ING1 present in thesample is measured indirectly by measuring the amount of a known, added(exogenous) antigen displaced (competed away) from an anti-antigenantibody by the unknown antigen present in a sample. In one competitiveassay, a known amount of antigen is added to a sample and the sample isthen contacted with an antibody that specifically binds to the antigen.The amount of exogenous antigen bound to the antibody is inverselyproportional to the concentration of antigen present in the sample. In aparticularly preferred embodiment, the antibody is immobilized on asolid substrate. The amount of antigen bound to the antibody may bedetermined either by measuring the amount of antigen present in anantigen/antibody complex, or alternatively by measuring the amount ofremaining uncomplexed protein. The amount of antigen may be detected byproviding a labeled antigen molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay the known antigen is immobilized on a solid substrate. Aknown amount of anti-antigen antibody is added to the sample, and thesample is then contacted with the immobilized antigen. The amount ofanti-antigen antibody bound to the known immobilized antigen isinversely proportional to the amount of antigen present in the sample.Again, the amount of immunobilized antibody may be detected by detectingeither the immobilized fraction of antibody or the fraction of theantibody that remains in solution. Detection may be direct where theantibody is labeled or indirect by the subsequent addition of a labeledmoiety that specifically binds to the antibody as described above.

Cross-reactivity determinations

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations. For example, p33ING2 or p33ING1 proteinscan be immobilized to a solid support. Proteins are added to the assaythat compete for binding of the antisera to the immobilized antigen. Theability of the added protein to compete for binding of the antisera tothe immobilized protein is compared to the ability of antigen to competewith itself The percent crossreactivity for the above proteins iscalculated, using standard calculations. Those antisera with less than10% crossreactivity with the added proteins are selected and pooled. Thecross-reacting antibodies are optionally removed from the pooledantisera by immunoabsorption with the added proteins.

Furthermore, immunoassays in the competitive binding format can be usedto determine cross-reactivity of polyclonal anti-p33ING2 antibodies orp33ING1 antibodies for p33ING1 and p33ING2 proteins, respectively. Asdescribed above, p33ING2 protein can be immobilized to a solid support.p33ING1 protein is added to the assay, and the ability of p33ING1protein to compete for binding of the antisera to the immobilizedp33ING1 protein is compared to the ability of p33ING2 to compete withitself. Those antisera with less than 10% crossreactivity with p33ING1protein are selected and pooled. Such immunoassays provides antibodiesthat selectively bind to a p33ING2 polypeptide but do not bind to ap33ING1 polypeptide. Similarly, immunoassays in the competitive bindingformat can be used to select antibodies that selectively bind to ap33ING1 polypeptide, but do not bind to a p33ING2 polypeptide. See,e.g., FIG. 2.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second proteinthought to be perhaps an allele, interspecies homologs, or polymorphicvariant of p33ING2 or p33ING1, to the immunogen protein. In order tomake this comparison, the two proteins are each assayed at a wide rangeof concentrations and the amount of each protein required to inhibit 50%of the binding of the antisera to the immobilized protein is determined.If the amount of the second protein required to inhibit 50% of bindingis less than 10 times the amount of the first protein that is requiredto inhibit 50% of binding, then the second protein is said tospecifically bind to the polyclonal antibodies generated to theimmunogen of choice.

Other assay formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of p33ING2 or p33ING1 in the sample. The technique generallycomprises separating sample proteins by gel electrophoresis on the basisof molecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind p33ING2. The anti-antigen antibodies specificallybind to the antigen on the solid support. These antibodies may bedirectly labeled or alternatively may be subsequently detected usinglabeled antibodies (e.g., labeled sheep anti-mouse antibodies) thatspecifically bind to the anti-antigen antibodies.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34-41 (1986)).

Reduction of non-specific binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly where theassay involves an antigen or antibody immobilized on a solid substrate,it is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²p), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecules (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize a specificprotein, or secondary antibodies that recognize antibodies to thespecific protein.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidotases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

VI. Assays for Measuring Changes in p33ING2 Regulated Cell Growth

P33ING2 and its alleles, interspecies homologs, and polymorphic variantsparticipate in regulation of cell proliferation and tumor suppression.Therefore, expression of p33ING2 and its alleles, interspecies homologs,and polymorphic variants in host cells would inhibit cell proliferationand suppress tumor formation. On the other hand, expression of p33ING2mutants in a cell could lead to abnormal cell proliferation and loss oftumor suppressor phenotypes. Finally, compounds that activate or inhibitp33ING2 would indirectly affect regulation of cellular proliferation andtumor suppression. Any of these changes in cell growth can be assessedby using a variety of in vitro and in vivo assays, e.g., ability to growon soft agar, changes in contact inhibition and density limitation ofgrowth, changes in growth factor or serum dependence, changes in thelevel of tumor specific markers, changes in invasiveness into Matrigel,changes in apoptosis, changes in cell cycle pattern, changes in tumorgrowth in vivo, such as in transgenic mice, etc. Furthermore, theseassays can be used to screen for activators, inhibitors, and modulatorsof p33ING2. Such activators, inhibitors, and modulators of p33ING2 canthen be used to modulate p33ING2 expression in tumor cells or abnormalproliferative cells.

A. Assays for changes in cell growth by expression of p33ING2 constructs

One or more of the following assays can be used to identify p33ING2constructs which are capable of regulating cell proliferation and tumorsuppression. The phrase “p33ING2 constructs” can refer to any of p33ING2and its alleles, interspecies homologs, polymorphic variants andmutants. Functional p33ING2 constructs identified by the followingassays can then be used in, e.g., gene therapy to inhibit abnormalcellular proliferation and transformation.

Soft agar growth or colony formation in suspension

Normal cells require a solid substrate to attach and grow. When thecells are transformed, they lose this phenotype and grow detached fromthe substrate. For example, transformed cells can grow in stirredsuspension culture or suspended in semi-solid media, such as semi-solidor soft agar. The transformed cells, when transfected with tumorsuppressor genes, regenerate normal phenotype and require a solidsubstrate to attach and grow.

Soft agar growth or colony formation in suspension assays can be used toidentify p33ING2 constructs, which when expressed in host cells, inhibitabnormal cellular proliferation and transformation. Typically,transformed host cells (e.g., cells that grow on soft agar) are used inthis assay. For example, RKO or HCT116 cell lines can be used.Expression of a tumor suppressor gene in these transformed host cellswould reduce or eliminate the host cells' ability to grow in stirredsuspension culture or suspended in semi-solid media, such as semi-solidor soft. This is because the host cells would regenerate anchoragedependence of normal cells, and therefore require a solid substrate togrow. Therefore, this assay can be used to identify p33ING2 constructsthat encode a functional tumor suppressor. Once identified, such p33ING2constructs can be used in a number of diagnostic or therapeutic methods,e.g., in gene therapy to inhibit abnormal cellular proliferation andtransformation.

Techniques for soft agar growth or colony formation in suspension assaysare described in Freshney, Culture of Animal Cells a Manual of BasicTechnique, 3^(rd) ed., Wiley-Liss, New York (1994), herein incorporatedby reference. See also, the methods section of Garkavtsev et al. (1996),supra, herein incorporated by reference.

Contact inhibition and density limitation of growth

Normal cells typically grow in a flat and organized pattern in a petridish until they touch other cells. When the cells touch one another,they are contact inhibited and stop growing. When cells are transformed,however, the cells are not contact inhibited and continue to grow tohigh densities in disorganized foci. Thus, the transformed cells grow toa higher saturation density than normal cells. This can be detectedmorphologically by the formation of a disoriented monolayer of cells orrounded cells in foci within the regular pattern of normal surroundingcells. Alternatively, labeling index with [³H]-thymidine at saturationdensity can be used to measure density limitation of growth. SeeFreshney (1994), supra. The transformed cells, when transfected withtumor suppressor genes, regenerate a normal phenotype and become contactinhibited and would grow to a lower density.

Contact inhibition and density limitation of growth assays can be usedto identify p33ING2 constructs which are capable of inhibiting abnormalproliferation and transformation in host cells. Typically, transformedhost cells (e.g., cells that are not contact inhibited) are used in thisassay. For example, RKO or HCT116 cell lines can be used. Expression ofa tumor suppressor gene in these transformed host cells would result incells which are contact inhibited and grow to a lower saturation densitythan the transformed cells. Therefore, this assay can be used toidentify p33ING2 constructs which function as a tumor suppressor. Onceidentified, such p33ING2 constructs can be used, e.g., in gene therapyto inhibit abnormal cellular proliferation and transformation.

In this assay, labeling index with [³H]-thymidine at saturation densityis a preferred method of measuring density limitation of growth.Transformed host cells are transfected with a p33ING2 construct and aregrown for 24 hours at saturation density in non-limiting mediumconditions. The percentage of cells labeling with [³H]-thymidine isdetermined autoradiogrpahically. See, Freshney (1994), supra. The hostcells expressing a functional p33ING2 construct would give arise to alower labeling index compared to control (e.g., transformed host cellstransfected with a vector lacking an insert).

Growth factor or serum dependence

Growth factor or serum dependence can be used as an assay to identifyfunctional p33ING2 constructs. Transformed cells have a lower serumdependence than their normal counterparts (see, e.g., Temin, J. Natl.Cancer Insti. 37:167-175 (1966); Eagle et al., J. Exp. Med. 131:836-879(1970)); Freshney, supra. This is in part due to release of variousgrowth factors by the transformed cells. When a tumor suppressor gene istransfected and expressed in these transformed cells, the cells wouldreacquire serum dependence and would release growth factors at a lowerlevel. Therefore, this assay can be used to identify p33ING2 constructswhich encode functional tumor suppressor. Growth factor or serumdependence of transformed host cells which are transfected with ap33ING2 construct can be compared with that of control (e.g.,transformed host cells which are transfected with a vector withoutinsert). Host cells expressing a functional p33ING2 would exhibit anincrease in growth factor and serum dependence compared to control.

Tumor specific markers levels

Tumor cells release an increased amount of certain factors (hereinafter“tumor specific markers”) than their normal counterparts. For example,plasminogen activator (PA) is released from human glioma at a higherlevel than from normal brain cells (see, e.g., Gullino, Angiogenesis,tumor vascularization, and potential interference with tumor growth. InMihich (ed.): “Biological Responses in Cancer.” New York, AcademicPress, pp. 178-184 (1985)). Similarly, Tumor angiogenesis factor (TAF)is released at a higher level in tumor cells than their normalcounterparts. See, e.g., Folkman, Angiogenesis and cancer, Sem CancerBiol. (1992)).

Tumor specific markers can be assayed for to identify p33ING2constructs, which when expressed, decrease the level of release of thesemarkers from host cells. Typically, transformed or tumorigenic hostcells are used. Expression of a tumor suppressor gene in these hostcells would reduce or eliminate the release of tumor specific markersfrom these cells. Therefore, this assay can be used to identify p33ING2constructs that encode a functional tumor suppressor.

Various techniques which measure the release of these factors aredescribed in Freshney (1994), supra. Also, see, Unkless et al. , J.Biol. Chem. 249:4295-4305 (1974); Strickland & Beers, J. Biol. Chem.251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305-312 (1980);Gulino, Angiogenesis, tumor vascularization, and potential interferencewith tumor growth. In Mihich, E. (ed): “Biological Responses in Cancer.”New York, Plenum (1985); Freshney Anticancer Res. 5:111-130 (1985).

Invasiveness into Matrigel

The degree of invasiveness into Matrigel or some other extracellularmatrix constituent can be used as an assay to identify p33ING2constructs which are capable of inhibiting abnormal cell proliferationand tumor growth. Tumor cells exhibit a good correlation betweenmalignancy and invasiveness of cells into Matrigel or some otherextracellular matrix constituent. In this assay, tumorigenic cells aretypically used as host cells. Expression of a tumor suppressor gene inthese host cells would decrease invasiveness of the host cells.Therefore, functional p33ING2 constructs can be identified by measuringchanges in the level of invasiveness between the host cells before andafter the introduction of p33ING2 constructs. If a p33ING2 constructfunctions as a tumor suppressor, its expression in tumorigenic hostcells would decrease invasiveness.

Techniques described in Freshney (1994), supra, can be used. Briefly,the level of invasion of host cells can be measured by using filterscoated with Matrigel or some other extracellular matrix constituent.Penetration into the gel, or through to the distal side of the filter,is rated as invasiveness, and rated histologically by number of cellsand distance moved, or by prelabeling the cells with ¹²⁵I and countingthe radioactivity on the distal side of the filter or bottom of thedish. See, e.g. Freshney (1984), supra.

Apoptosis analysis

Apoptosis analysis can be used as an assay to identify functionalp33ING2 constructs. p33ING2 expression or overexpression causesapoptosis (see Example IX below). In this assay, cell lines, such as RKOor HCT116, can be used to screen p33ING2 constructs which encode afunctional tumor suppressor. Cells are transfected with a putativep33ING2 construct. The cells can be co-transfected with a constructcomprising a marker gene, such as a gene that encodes green fluorescentprotein. Alternatively, a single construct comprising a putative p33ING2gene and a marker gene can be transfected into cells. Overexpression ofa p33ING2 gene that encodes a functional tumor suppressor would causeapoptosis. Not wishing to be bound by a theory, exogenous expression ofa tumor suppressor can decrease cell proliferation by causing a cellcycle arrest and by increasing cell death. The apoptotic change can bedetermined using methods known in the art, such as DAPI staining andTUNEL assay using fluorescent microscope. For TUNEL assay, commerciallyavailable kit can be used (e.g., Fluorescein FragEL DNA FragmentationDetection Kit (Oncogene Research Products, Cat.#QIA39)+Tetramethyl-rhodamine-5-dUTP (Roche, Cat #1534 378)). Cellsexpressing a functional p33ING2 would exhibit an increased apoptosiscompared to control (e.g., a cell transfected with a vector without ap33ING2 gene insert).

G₀/G₁ cell cycle arrest analysis

G₀/G₁ cell cycle arrest can be used as an assay to identify functionalp33ING2 constructs. p33ING2 expression or overexpression causes G1 cellcycle arrest (see Example IX below). In this assay, cell lines, such asRKO or HCT116, can be used to screen p33ING2 constructs which encode afunctional tumor suppressor. Cells are transfected with a putativep33ING2 construct. The cells can be co-transfected with a constructcomprising a marker gene, such as a gene that encodes green fluorescentprotein. Alternatively, a single construct comprising a putative p33ING2gene and a marker gene can be transfected into cells. Expression oroverexpression of a p33ING2 gene that encodes a functional tumorsuppressor would cause G₀/G₁ cell cycle arrest (see, e.g., Example VII).Methods known in the art can be used to measure the degree of G₁ cellcycle arrest. For example, the propidium iodide signal can be used as ameasure for DNA content to determine cell cycle profiles on a flowcytometer. The percent of the cells in each cell cycle can becalculated. Cells expressing a functional p33ING2 would exhibit a highernumber of cells that are arrested in G₀/G₁ phase compared to control(e.g., transfected with a vector without a p33ING2 gene insert).

Tumor growth in vivo

Effects of p33ING2 on cell growth can be tested in transgenic orimmune-suppressed mice. Knock-out transgenic mice can be made, in whichthe endogenous p33ING2 gene is disrupted. Such knock-out mice can beused to study effects of p33ING2, e.g., as a cancer model, as a means ofassaying in vivo for compounds that modulate p33ING2, and to test theeffects of restoring a wildtype p33ING2 to a knock-out mice.

Knock-out transgenic mice can be made by insertion of a marker gene orother heterologous gene into the endogenous p33ING2 gene site in themouse genome via homologous recombination. Such mice can also be made bysubstituting the endogenous p33ING2 with a mutated version of p33ING2,or by mutating the endogenous p33ING2, e.g., by exposure to carcinogens.

A DNA construct is introduced into the nuclei of embryonic stem cells.Cells containing the newly engineered genetic lesion are injected into ahost mouse embryo, which is re-implanted into a recipient female. Someof these embryos develop into chimeric mice that possess germ cellspartially derived from the mutant cell line. Therefore, by breeding thechimeric mice it is possible to obtain a new line of mice containing theintroduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288(1989)). Chimeric targeted mice can be derived according to Hogan etal., Manipulating the Mouse Embryo: A Laboratory Manual, Cold SpringHarbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells:A Practical Approach, Robertson, ed., IRL Press, Washington, D.C.,(1987).

These knock-out mice can be used as hosts to test the effects of variousp33ING2 constructs on cell growth. These transgenic mice with theendogenous p33ING2 gene knocked out would develop abnormal cellproliferation and tumor growth. They can be used as hosts to test theeffects of various p33ING2 constructs on cell growth. For example,introduction of wildtype p33ING2 into these knock-out mice would inhibitabnormal cellular proliferation and suppress tumor growth.

Alternatively, various immun-suppressed or immune-deficient host animalscan be used. For example, genetically athymic “nude” mouse (see, e.g.,Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), a SCID mouse, athymectomized mouse, or an irradiated mouse (see, e.g., Bradley et al.,Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer 41:52 (1980))can be used as a host. Transplantable tumor cells (typically about 10⁶cells) injected into isogenic hosts will produce invasive tumors in ahigh proportions of cases, while normal cells of similar origin willnot. In hosts which developed invasive tumors, cells expressing ap33ING2 construct are injected subcutaneously. After a suitable lengthof time, preferably 4-8 weeks, tumor growth is measured (e.g., by volumeor by its two largest dimensions) and compared to the control. Tumorsthat have statistically significant reduction (using, e.g., Student's Ttest) are said to have inhibited growth. Using reduction of tumor sizeas an assay, functional p33ING2 constructs which are capable ofinhibiting abnormal cell proliferation can be identified. This model canalso be used to identify mutant versions of p33ING2.

B. Assays for compounds that modulate p33ING2

P33ING2 and its alleles, interspecies homologs, and polymorphic variantsparticipate in regulation of cell proliferation and tumor suppression.Mutations in these genes, including null or missense mutations, cancause abnormal cell proliferation and tumor growth The activity ofp33ING2 polypeptides (wildtype or mutants) can be assessed using avariety of in vitro and in vivo assays measuring various parameters,e.g., cell growth on soft agar, contact inhibition and densitylimitation of growth, growth factor or serum dependence, tumor specificmarkers levels, invasiveness into Matrigel, tumor growth in vivo,transgenic mice, p33ING2 protein or mRNA levels, transcriptionalactivation or repression of a reporter gene, apoptosis analysis, G₀/G₁cell cycle arrest, and the like. Such assays can also be used to screenfor activators, inhibitors, and modulators of wildtype and mutantp33ING2. Such activators, inhibitors, and modulators are useful ininhibiting tumor growth and modulating cell proliferation. Compoundsidentified using the assays of the invention are useful as therapeuticsfor treatment of cancer and other diseases involving cellularhyperproliferation.

Biologically active or inactivated p33ING2 polypeptides, eitherrecombinants or naturally occurring, are used to screen activators,inhibitors, or modulators of tumor suppression and cell proliferation.The p33ING2 polypeptides can be recombinantly expressed in a cell,naturally expressed in a cell, recombinantly or naturally expressed incells transplanted into an animal, or recombinantly or naturallyexpressed in a transgenic animal. Modulation is tested using one of thein vitro or in vivo assays described in herein in part A.

Cells that have wildtype p33ING2, p33ING2 null mutations, p33ING2missense mutations, or inactivation of p33ING2 are used in the assays ofthe invention, both in vitro and in vivo. Preferably, human cells areused. Cell lines can also be created or isolated from tumors that havemutant p33ING2. Optionally, the cells can be transfected with anexogenous p33ING2 gene operably linked to a constitutive promoter, toprovide higher levels of p33ING2 expression. Alternatively, endogenousp33ING2 levels can be examined. The cells can be treated to inducep33ING2 expression. The cells can be immobilized, be in solution, beinjected into an animal, or be naturally occurring in a transgenic ornon-transgenic animal.

Samples or assays that are treated with a test compound whichpotentially activates, inhibits, or modulates p33ING2 are compared tocontrol samples that are not treated with the test compound, to examinethe extent of modulation. Generally, the compounds to be tested arepresent in the range from 0.1 mM to 10 mM. Control samples (untreatedwith activators, inhibitors, or modulators) are assigned relativep33ING2 activity value of 100%. Inhibition of p33ING2 is achieved whenthe p33ING2 activity value relative to the control is about 90% (e.g.,10% less than the control), optionally 80% or less, 70% or less, 60% orless, 50% or less, 40% or less, or 25-0%. Activation of p33ING2 isachieved when the p33ING2 activity value relative to the control is 110%or more (e.g., at least 10% more than the control), optionally 120%,130%, 140%, 150% or more, 200-500% or more, 1000-3000% or more.

The effects of the test compounds upon the function of the p33ING2polypeptides can be measured by examining any of the parametersdescribed above. For example, parameters such as ability to grow on softagar, contact inhibition and density limitation of growth, growth factoror serum dependence, tumor specific markers levels, invasiveness intoMatrigel, apoptosis, G₀/G₁ cell cycle arrest, tumor growth in vivo,transgenic mice and the like, can be measured. Furthermore, the effectsof the test compounds on p33ING2 protein or mRNA levels, transcriptionalactivation or repression of a reporter gene can be measured. In eachassay, cells expressing p33ING2 are contacted with a test compound andincubated for a suitable amount of time, e.g., from 0.5 to 48 hours.Then, parameters such as those described above are compared to thoseproduced by control cells untreated with the test compound.

In one embodiment, the effect of test compounds upon the function ofp33ING2 can be determined by comparing the level of p33ING2 protein ormRNA in treated samples and control samples. The level of p33ING2protein is measured using immunoassays such as western blotting, ELISAand the like with a p33ING2 specific antibody. For measurement of mRNA,amplification, e.g., using PCR, LCR, or hybridization assays, e.g.,northern hybridization, RNase protection, dot blotting, are preferred.The level of protein or mRNA is detected using directly or indirectlylabeled detection agents, e.g., fluorescently or radioactively labelednucleic acids, radioactively or enzymatically labeled antibodies, andthe like, as described herein.

Alternatively, a reporter gene system can be devised using the p33ING2promoter operably linked to a reporter gene such as luciferase, greenfluorescent protein, CAT, or β-gal. After treatment with a potentialp33ING2 modulator, the amount of reporter gene transcription,translation, or activity is measured according to standard techniquesknown to those of skill in the art.

In another embodiment, the effects of test compounds on p33ING2 activityis performed in vivo. In this assay, cultured cells that are expressinga wildtype or mutant p33ING2 (e.g., a null or missense mutation) areinjected subcutaneously into an immune compromised mouse such as anathymic mouse, an irradiated mouse, or a SCID mouse. P33ING2 modulatorsare administered to the mouse, e.g., a chemical ligand library. After asuitable length of time, preferably 4-8 weeks, tumor growth is measured,e.g., by volume or by its two largest dimensions, and compared to thecontrol. Tumors that have statistically significant reduction (using,e.g., Student's T test) are said to have inhibited growth.Alternatively, the extent of tumor neovascularization can also bemeasured. Immunoassays using endothelial cell specific antibodies areused to stain for vascularization of the tumor and the number of vesselsin the tumor. Tumors that have a statistically significant reduction inthe number of vessels (using, e.g., Student's T test) are said to haveinhibited neovascularization.

Alternatively, transgenic mice with the endogenous p33ING2 gene knockedout can be used in an assay to screen for compounds which modulate thep33ING2 activity. As described in part A, knock-out transgenic mice canbe made, in which the endogenous p33ING2 gene is disrupted, e.g., byreplacing it with a marker gene. A transgenic mouse that is heterozygousor homozygous for integrated transgenes that have functionally disruptedthe endogenous p33ING2 gene can be used as a sensitive in vivo screeningassay for p33ING2 ligands and modulators of p33ING2 activity.

C Modulators

The compounds tested as modulators of p33ING2 can be any small chemicalcompound, or a biological entity, such as a protein, sugar, nucleic acidor lipid. Alternatively, modulators can be genetically altered versionsof p33ING2. For example, an antisense construct of p33ING2 can be usedas a modulator.

Typically, test compounds will be small chemical molecules and peptides.Essentially any chemical compound can be used as a potential modulatoror ligand in the assays of the invention, although most often compoundscan be dissolved in aqueous or organic (especially DMSO-based) solutionsare used. The assays are designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource to assays, which are typically run in parallel (e.g., inmicrotiter formats on microtiter plates in robotic assays). It will beappreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as bydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho etal., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN,January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton,Pa., Martek Biosciences, Columbia, Md., etc.).

D. Solid state and soluble high throughput assays

In one embodiment the invention provide soluble assays using moleculessuch as a domain such as ligand binding domain, an active site, etc.; adomain that is covalently linked to a heterologous protein to create achimeric molecule; p33ING2; a cell or tissue expressing p33ING2, eithernaturally occurring or recombinant. In another embodiment, the inventionprovides solid phase based in vitro assays in a high throughput format,where the domain, chimeric molecule, p33ING2, or cell or tissueexpressing p33ING2 is attached to a solid phase substrate.

In the high throughput assays of the invention, it is possible to screenup to several thousand different modulators or ligands in a single day.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100-1500 differentcompounds. It is possible to assay several different plates per day;assay screens for up to about 6,000-20,000 different compounds ispossible using the integrated systems of the invention.

The molecule of interest can be bound to the solid state component,directly or indirectly, via covalent or non covalent linkage, e.g., viaa tag. The tag can be any of a variety of components. In general, amolecule which binds the tag (a tag binder) is fixed to a solid support,and the tagged molecule of interest is attached to the solid support byinteraction of the tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.)Antibodies to molecules with natural binders such as biotin are alsowidely available and appropriate tag binders; see, SIGMA Immunochemical1998 catalogue SIGMA, St. Louis Mo.

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherein family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g. which mediate the effects of various smallligands, including steroids, thyroid hormone, retinoids and vitamin D;peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclicpolymer configurations), oligosaccharides, proteins, phospholipids andantibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimincs, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly gly sequencesof between about 5 and 200 amino acids. Such flexible linkers are knownto persons of skill in the art. For example, poly(ethylene glycol)linkers are available from Shearwater Polymers, Inc. Huntsville, Ala.These linkers optionally have amide linkages, sulfhydryl linkages, orheterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is Well described in the literature. See, e.g., Merrifield, J.Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)(describing synthesis of solid phase components on pins); Frank &Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719(1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

E. Computer-based assays

Yet another assay for compounds that modulate p33ING2 activity involvescomputer assisted drug design, in which a computer system is used togenerate a three-dimensional structure of p33ING2 based on thestructural information encoded by the amino acid sequence. The inputamino acid sequence interacts directly and actively with apreestablished algorithm in a computer program to yield secondary,tertiary, and quaternary structural models of the protein. The models ofthe protein structure are then examined to identify regions of thestructure that have the ability to bind, e.g., ligands. These regionsare then used to identify ligands that bind to the protein.

The three-dimensional structural model of the protein is generated byentering p33ING2 amino acid sequences of at least 10 amino acid residuesor corresponding nucleic acid sequences encoding a p33ING2 polypeptideinto the computer system. The amino acid sequence of the polypeptide orthe nucleic acid encoding the polypeptide is selected from the groupconsisting of SEQ ID NO:1 or SEQ ID NO:2, and conservatively modifiedversions thereof. The amino acid sequence represents the primarysequence or subsequence of the protein, which encodes the structuralinformation of the protein. At least 10 residues of the amino acidsequence (or a nucleotide sequence encoding 10 amino acids) are enteredinto the computer system from computer keyboards, computer readablesubstrates that include, but are not limited to, electronic storagemedia (e.g., magnetic diskettes, tapes, cartridges, and chips), opticalmedia (e.g., CD ROM), information distributed by internet sites, and byRAM. The three-dimensional structural model of the protein is thengenerated by the interaction of the amino acid sequence and the computersystem, using software known to those of skill in the art. Thethree-dimensional structural model of the protein can be saved to acomputer readable form and be used for further analysis (e.g.,identifying potential ligand binding regions of the protein andscreening for mutations, alleles and interspecies homologs of the gene).

The amino acid sequence represents a primary structure that encodes theinformation necessary to form the secondary, tertiary and quaternarystructure of the protein of interest. The software looks at certainparameters encoded by the primary sequence to generate the structuralmodel. These parameters are referred to as “energy terms,” and primarilyinclude electrostatic potentials, hydrophobic potentials, solventaccessible surfaces, and hydrogen bonding. Secondary energy termsinclude van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

The tertiary structure of the protein encoded by the secondary structureis then formed on the basis of the energy terms of the secondarystructure. The user at this point can enter additional variables such aswhether the protein is membrane bound or soluble, its location in thebody, and its cellular location, e.g., cytoplasmic, surface, or nuclear.These variables along with the energy terms of the secondary structureare used to form the model of the tertiary structure. In modeling thetertiary structure, the computer program matches hydrophobic faces ofsecondary structure with like, and hydrophilic faces of secondarystructure with like.

Once the structure has been generated, potential ligand binding regionsare identified by the computer system. Three-dimensional structures forpotential ligands are generated by entering amino acid or nucleotidesequences or chemical formulas of compounds, as described above. Thethree-dimensional structure of the potential ligand is then compared tothat of the p33ING2 protein to identify ligands that bind to p33ING2.Binding affinity between the protein and ligands is determined usingenergy terms to determine which ligands have an enhanced probability ofbinding to the protein. The results, such as three-dimensionalstructures for potential ligands and binding affinity of ligands, canalso be saved to a computer readable form and can be used for furtheranalysis (e.g., generating a three dimensional model of mutated proteinshaving an altered binding affinity for a ligand).

Computer systems are also used to screen for mutations, polymorphicvariants, alleles and interspecies homologs of p33ING2 genes. Suchmutations can be associated with disease states or genetic traits. Asdescribed above, high density oligonucleotide arrays (GeneChip™) andrelated technology can also be used to screen for mutations, polymorphicvariants, alleles and interspecies homologs. Once the variants areidentified, diagnostic assays can be used to identify patients havingsuch mutated genes. Identification of the mutated p33ING2 genes involvesreceiving input of a first nucleic acid or amino acid sequence encodingselected from the group consisting of SEQ ID NO:2, or SEQ ID NO:1, andconservatively modified versions thereof. The sequence is entered intothe computer system as described above and then saved to a computerreadable form. The first nucleic acid or amino acid sequence is thencompared to a second nucleic acid or amino acid sequence that hassubstantial identity to the first sequence. The second sequence isentered into the computer system in the manner described above. Once thefirst and second sequences are compared, nucleotide or amino aciddifferences between the sequences are identified. Such sequences canrepresent allelic differences in p33ING2 genes, and mutations associatedwith disease states and genetic traits.

VII. Gene Therapy

The present invention provides the nucleic acids of p33ING2 for thetransfection of cells in vitro and in vivo. These nucleic acids can beinserted into any of a number of well known vectors for the transfectionof target cells and organisms as described below. The nucleic acids aretransfected into cells, ex vivo or in vivo, through the interaction ofthe vector and the target cell. The nucleic acids encoding p33ING2,under the control of a promoter, then expresses a p33ING2 of the presentinvention, thereby mitigating the effects of absent, partialinactivation, or abnormal expression of the p33ING2 gene.

Such gene therapy procedures have been used to correct acquired andinherited genetic defects, cancer, and viral infection in a number ofcontexts. The ability to express artificial genes in humans facilitatesthe prevention and/or cure of many important human diseases, includingmany diseases which are not amenable to treatment by other therapies(for a review of gene therapy procedures, see Anderson, Science256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani &Caskey, TIBTECH 11:162-166 (1993); Mulligan, Science 926-932 (1993);Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992);Van Brunt, Biotechnology 6(10):1149-1154 (1998); Vigne, RestorativeNeurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, BritishMedical Bulletin 51(l):31-44 (1995); Haddada et al., in Current Topicsin Microbiology and Immunology (Doerfler & Böhm eds., 1995); and Yu etal., Gene Therapy 1:13-26 (1994)).

Delivery of the gene or genetic material into the cell is the firstcritical step in gene therapy treatment of disease. A large number ofdelivery methods are well known to those of skill in the art.Preferably, the nucleic acids are administered for in vivo or ex vivogene therapy uses. Non-viral vector delivery systems include DNAplasmids, naked nucleic acid, and nucleic acid complexed with a deliveryvehicle such as a liposome. Viral vector delivery systems include DNAand RNA viruses, which have either episomal or integrated genomes afterdelivery to the cell. For a review of gene therapy procedures, seeAnderson, Science 256:808-813 (1992); Nabel & Feigner, TIBTECH11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon,TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt,Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology andNeuroscience 8:35-36 (1995); Kremer & Perricaudet, British MedicalBulletin 51(1):31-44 (1995); Haddada et al., in Current Topics inMicrobiology and Immunology Doerfler and Böhm (eds) (1995); and Yu etal., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection,microinjection, biolistics, virosomes, liposomes, immunoliposomes,polycation or lipid:nucleic acid conjugates, naked DNA, artificialvirions, and agent-enhanced uptake of DNA. Lipofection is described in,e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) andlipofection reagents are sold commercially (e.g., Transfectam™ andLipofectin™). Cationic and neutral lipids that are suitable forefficient receptor-recognition lipofection of polynucleotides includethose of Feigner, WO 91/17424, WO 91/16024. Delivery can be to cells (exvivo administration) or target tissues (in vivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids take advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro and the modified cellsare administered to patients (ex vivo). Conventional viral based systemsfor the delivery of nucleic acids could include retroviral, lentivirus,adenoviral, adeno-associated and herpes simplex virus vectors for genetransfer. Viral vectors are currently the most efficient and versatilemethod of gene transfer in target cells and tissues. Integration in thehost genome is possible with the retrovirus, lentivirus, andadeno-associated virus gene transfer methods, often resulting in longterm expression of the inserted transgene. Additionally, hightransduction efficiencies have been observed in many different celltypes and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vector that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SIV), human immuno deficiency virus(HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications where transient expression of the nucleic acid ispreferred, adenoviral based systems are typically used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors are also used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J.Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, Proc. Natl. Acad. Sci. U.S.A. 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

In particular, at least six viral vector approaches are currentlyavailable for gene transfer in clinical trials, with retroviral vectorsby far the most frequently used system. All of these viral vectorsutilize approaches that involve complementation of defective vectors bygenes inserted into helper cell lines to generate the transducing agent.

pLASN and MFG-S are examples are retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., Proc. Natl. Acad. Sci.U.S.A. 94:22 12133-12138 (1997)). PA317/pLASN was the first therapeuticvector used in a gene therapy trial. (Blaese et al., Science 270:475-480(1995)). Transduction efficiencies of 50% or greater have been observedfor MFG-S packaged vectors. (Ellem et at, Immunol Immunother.44(1):10-20 (1997); Dranoff et al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)).

Replication-deficient recombinant adenoviral vectors (Ad) arepredominantly used transient expression gene therapy, because they canbe produced at high titer and they readily infect a number of differentcell types. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and E3 genes; subsequently the replicationdefector vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiply types oftissues in vivo, including nondividing, differentiated cells such asthose found in the liver, kidney and muscle system tissues. ConventionalAd vectors have a large carrying capacity. An example of the use of anAd vector in a clinical trial involved polynucleotide therapy forantitumor immunization with intramuscular injection (Sterman et al.,Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use ofadenovirus vectors for gene transfer in clinical trials includeRosenecker et al., Infection 24:1 5-10 (1996); Sterman et al., Hum. GeneTher. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18(1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al.,Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089(1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by producer cell linethat packages a nucleic acid vector into a viral particle. The vectorstypically contain the minimal viral sequences required for packaging andsubsequent integration into a host, other viral sequences being replacedby an expression cassette for the protein to be expressed. The missingviral functions are supplied in trans by the packaging cell line. Forexample, AAV vectors used in gene therapy typically only possess ITRsequences from the AAV genome which are required for packaging andintegration into the host genome. Viral DNA is packaged in a cell line,which contains a helper plasmid encoding the other AAV genes, namely repand cap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. A viral vector is typically modified to have specificityfor a given cell type by expressing a ligand as a fusion protein with aviral coat protein on the viruses outer surface. The ligand is chosen tohave affinity for a receptor known to be present on the cell type ofinterest. For example, Han et al., Proc. Natl. Acad. Sci. U.S.A.92:9747-9751 (1995), reported that Moloney murine leukemia virus can bemodified to express human heregulin fused to gp70, and the recombinantvirus infects certain human breast cancer cells expressing humanepidermal growth factor receptor. This principle can be extended toother pairs of virus expressing a ligand fusion protein and target cellexpressing a receptor. For example, filamentous phage can be engineeredto display antibody fragments (e.g., Fab or Fv) having specific bindingaffinity for virtually any chosen cellular receptor. Although the abovedescription applies primarily to viral vectors, the same principles canbe applied to nonviral vectors. Such vectors can be engineered tocontain specific uptake sequences thought to favor uptake by specifictarget cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with a nucleicacid (gene or cDNA), and re-infused back into the subject organism(e.g., patient). Various cell types suitable for ex vivo transfectionare well known to those of skill in the art (see, e.g., Freshney et al.,Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) andthe references cited therein for a discussion of how to isolate andculture cells from patients).

In one embodiment, stem cells are used in ex vivo procedures for celltransfection and gene therapy. The advantage to using stem cells is thatthey can be differentiated into other cell types in vitro, or can beintroduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+ cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-α are known (see Inaba et al., J. Exp. Med.176:1693-1702 (1992)).

Stem cells are isolated for transduction and differentiation using knownmethods. For example, stem cells are isolated from bone marrow cells bypanning the bone marrow cells with antibodies which bind unwanted cells,such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1(granulocytes), and Iad (differentiated antigen presenting cells) (seeInaba et al., J. Exp. Med. 176:1693-1702 (1992)).

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic nucleic acids can be also administered directly to theorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells, as describedbelow. The nucleic acids are administered in any suitable manner,preferably with pharmaceutically acceptable carriers. Suitable methodsof administering such nucleic acids are available and well known tothose of skill in the art, and, although more than one route can be usedto administer a particular composition, a particular route can oftenprovide a more immediate and more effective reaction than another route(see Proc. Natl. Acad. Sci. U.S.A. 81:6466-6470 (1984); and Samulski etal., J. Virol. 63:03822-3828 (1989)). In particular, at least six viralvector approaches are currently available for gene transfer in clinicaltrials, with retroviral vectors by far the most frequently used system.All of these viral vectors utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

VIII. Pharmaceutical Compositions and Administration

p33ING2 nucleic acid, protein, and modulators of p33ING2 can beadministered directly to the patient for inhibition of cancer, tumor, orprecancer cells in vivo. Administration is by any of the routes normallyused for introducing a compound into ultimate contact with the tissue tobe treated. The compounds are administered in any suitable manner,preferably with pharmaceutically acceptable carriers. Suitable methodsof administering such compounds are available and well known to those ofskill in the art, and, although more than one route can be used toadminister a particular composition, a particular route can oftenprovide a more immediate and more effective reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention (see, e.g., Remington's Pharmaceutical Sciences,17^(th) ed. 1985)). For example, if in vivo delivery of a biologicallyactive p33ING2 protein is desired, the methods described in Schwarze etal. (see Science 285:1569-1572 (1999)) can be used.

The compounds (nucleic acids, proteins, and modulators), alone or incombination with other suitable components, can be made into aerosolformulations (i.e., they can be “nebulized”) to be administered viainhalation. Aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intravenous, intramuscular, intradermal, and subcutaneousroutes, include aqueous and non-aqueous, isotonic sterile injectionsolutions, which can contain antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient, and aqueous and non-aqueous sterile suspensions thatcan include suspending agents, solubilizers, thickening agents,stabilizers, and preservatives. In the practice of this invention,compositions can be administered, for example, by intravenous infusion,orally, topically, intraperitoneally, intravesically or intrathecally.The formulations of compounds can be presented in unit-dose ormulti-dose sealed containers, such as ampules and vials. Injectionsolutions and suspensions can be prepared from sterile powders,granules, and tablets of the kind previously described.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular compound employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular compound or vector in a particularpatient.

In determining the effective amount of the modulator to be administeredin the treatment or prophylaxis of cancer, the physician evaluatescirculating plasma levels of the modulator, modulator toxicities,progression of the disease, and the production of anti-modulatorantibodies. In general, the dose equivalent of a modulator is from about1 ng/kg to 10 mg/kg for a typical patient. Administration of compoundsis well known to those of skill in the art (see, e.g., Bansinath et al.,Neurochem Res. 18:1063-1066 (1993); Iwasaki et al., Jpn. J. Cancer Res.88:861-866 (1997); Tabrizi-Rad et al., Br. J. Pharmacol. 111:394-396(1994)).

For administration, modulators of the present invention can beadministered at a rate determined by the LD-50 of the modulator, and theside-effects of the inhibitor at various concentrations, as applied tothe mass and overall health of the patient. Administration can beaccomplished via single or divided doses.

IX. Diagnostics and Kits

The present invention also provides methods for detection of p33ING2(either wildtype or mutant). For example, kits are provided that containp33ING2 specific reagents that specifically hybridize to p33ING2 nucleicacid, such as specific probes and primers, and p33ING2 specific reagentsthat specifically bind to the protein of choice, e.g., antibodies. Themethods, kits, and the assays described herein can be used foridentification of modulators of p33ING2, or for diagnosing patients withmutations in p33ING2.

Nucleic acid assays for the presence of p33ING2 DNA and RNA in a sampleinclude numerous techniques are known to those skilled in the art. Inparticular, p33ING2 specific reagents (e.g., p33ING2-specific primers ornucleic acid probes) can be used to distinguish between samples whichcontain p33ING2 nucleic acids and samples which contain p33ING1 nucleicacids. Techniques such as Southern analysis, Northern analysis, dotblots, RNase protection, high density oligonucleotide arrays, S1analysis, amplification techniques such as PCR and LCR, and in situhybridization can be used as assays. In in situ hybridization, forexample, the target nucleic acid is liberated from its cellularsurroundings in such as to be available for hybridization within thecell while preserving the cellular morphology for subsequentinterpretation and analysis. The following articles provide an overviewof the art of in situ hybridization: Singer et al., Biotechniques4:230-250 (1986); Haase et al., Methods in Virology, vol. VII, pp.189-226 (1984); and Nucleic Acid Hybridization: A Practical Approach(Hames et al., eds. 1987).

In addition, p33ING2 protein can be detected with the variousimmunoassay techniques described above, e.g., ELISA, western blotting,and the like. The test sample is typically compared to both a positivecontrol (e.g., a sample expressing recombinant p33ING2) and a negativecontrol. In particular, p33ING2 specific polyclonal and monoclonalantibodies or p33ING1 specific polyclonal and monoclonal antibodies canbe used as a diagnostic tool to distinguish between samples whichcontain p33ING2 antigens and samples which contain p33ING1 antigens.These polyclonal and monoclonal antibodies can also be used to determinethe amount of p33ING2 or p33ING1 in samples.

The present invention also provides for kits for screening formodulators of p33ING2. Such kits can be prepared from readily availablematerials and reagents. For example, such kits can comprise any one ormore of the following materials: p33ING2, reaction tubes, andinstructions for testing p33ING2 activity. Preferably, the kit containsbiologically active p33ING2. Furthermore, the kit may include a label orwritten instructions for the use of one or more of these reagents andmaterials in any of the assays described herein. A wide variety of kitsand components can be prepared according to the present invention,depending upon the intended user of the kit and the particular needs ofthe user.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example I Cloning and Expression of p33ING2

p33ING1 homologous sequences were found in a random cDNA sequencedatabase consisting of short partial sequences known as expressedsequence tags (ESTs) submitted in GenBank. Using primers designed basedon these EST sequences and using RT-PCR and 5′- and 3′-RACE methods,p33ING2 coding region (SEQ ID NO:2) from human placenta cDNA (CLONTECH)was isolated and subcloned into a plasmid.

To obtain a p33ING2 genomic sequence, a human PAC genomic library wasscreened with the p33ING2 cDNA sequence. Two clones were selected, oneof which included the p33ING2 genomic sequence (SEQ ID NO:7;exon1/intron). The genomic structure (exon/intron boundary sequence) wasdetermined by using human PAC genomic clones and “long distancesequence” method. The nucleic acid sequence of SEQ ID NO:10 isexon2/intron of p33ING2 genomic sequence.

Chromosomal localization of p33ING2 was determined using FISH(fluorescent in situ hybridization) analysis and human PAC genomic cloneincluding p33ING2 genome as a probe. It was determined that p33ING2 islocated at chromosome 4, at 4q35.

One human cancer cell line with a p33ING2 mutation, namely, HCT116, wasdiscovered. As shown in SEQ ID NO:6, it has a missense mutation at aminoacid position 153 (Arg to Ser).

Example II Cloning and Expression of p133ING1

The p33ING1 mRNA coding and amino acid sequences submitted in GenBank(Accession No. AF044076) had several mistakes. The correct sequence ofp33ING1 mRNA coding region was determined by using human placental cDNAand RT-PCR method.

A human PAC genomic library was screened by p33ING1 cDNA sequence. Twoclones were picked up which included p33ING1 genomic sequence. Thegenomic structure (exon/intron boundary sequence) was determined byusing the human PAC genomic clones and “long distance sequence” method.The sequence of mRNA coding sequence was also confirmed by the genomicDNA sequence.

Example III Antibodies to p33ING2 and p33ING1

Antibodies to p33ING2 and p33ING1 were synthesized using two uniquepeptides (KMP-1 from p33ING2 (see, e.g., SEQ ID NO:5) and KMP-2 fromp33ING1 (see, e.g., SEQ ID NO:9)). These peptides were purified by HPLC;peptide KLH conjugations were made; and rabbits were immunized by them.Antiserum was purified using peptide affinity column and specificity ofeach polyclonal antibody was analyzed by ELISA.

By ELISA (enzyme-linked immunosorbent assay) anti-p33ING2 polyclonalantibodies are reactive with recombinant GST-p33ING2 protein or itspeptide fragment KMP-1 (SEQ ID NO:5), but are not cross-reactive withrecombinant GST-p33ING1 protein or its peptide fragment KMP-2 (SEQ IDNO:9). Anti-p33ING1 polyclonal antibodies are reactive with recombinantGST-p33ING1 protein or its peptide fragment KMP-2, but are notcross-reactive with recombinant GST-p33ING2 protein or its peptidefragment.

As shown in FIG. 1, by Western blot analysis, anti-p33ING2 polyclonalantibodies are reactive with recombinant p33ING2 protein, but are notcross-reactive with recombinant p33ING1 protein. Anti-p33ING1 polyclonalantibodies are reactive with recombinant p33ING1 protein, but are notcross-reactive with recombinant p33ING2 protein.

Example IV Inhibition of Cell Proliferation

The colony formation assay was used to determine if p33ING2 inhibitscell growth of HCT116 cell line (human, hereditary non-polyposis coloncancer cell line, wt p53).

Mammalian expression vectors (with CMV promoter, Neomycin resistant)containing p33ING2 in sense orientation (pcDNA3-ING2) and in antisenseorientation (pcDNA3-AntiING2) were constructed. HCT116 cell lines weretransfected with the expression vectors. The transfected cells wereselected by Neomycin. The colony formation assay was used to test theeffect of p33ING2 and anti-p33ING2 expression in HCT116 cell lines.HCT116 cells transfected with pcDNA3-ING2 formed less colonies comparedto HCT116 cells transfected with pcDNA3-AntiING2 or HCT116 cellstransfected with pcDNA3 (without any inserts), demonstrating thatp33ING2 inhibits cell growth.

Example V Soft Agar Assay for Identifying Compounds that Modulatep33ING2

Wildtype or mutant p33ING2 is expressed in host cells to screencompounds that modulate anchorage dependence of host cells expressingp33ING2. This is achieved by using the method disclosed in Garkavtsev etal. (1996), supra, herein incorporated by reference. Non-tumorigenicimmortalized mouse mammary epithelial cells (NMuMG) are transfected withretrovirus produced from a vector containing p33ING2 in sense orantisense orientation, or a vector lacking insert (control). The softagar culture is comprised of two layers: an underlay (DMEM, 10% FCS,0.6% agar) and an overlay (DMEM, 10% FCS, 0.3% agar), 5×10⁴ cells areplated in soft agar in 10 cm plates are left at 37 ° C. for 6-7 weeksbefore being counted. The cells are incubated with a test compound for asuitable amount of time, e.g., for 0.5 to 48 hours, before countingcells. The amount of cells in the test sample is then compared tocontrol cells untreated with the compound.

Example VI p33ING2 Protein Induction by DNA Damage

Calu6 cells were treated with topoisomerase II inhibitor, etoposide(SIGMA, E-1383, 10 μg/ml). Etoposide can induce DNA damage (e.g.,double-strand DNA break). FIG. 2 shows the Western blot of p33ING1,p33ING2 and beta-actin (as control). The protein analysis indicated thatp33ING2 protein expression was induced by the treatment of Calu6 cellswith etoposide. However, p33ING1 protein expression was not induced byetopside.

Example VII p33ING1 or p33ING2 Can Induce G₁ Cell Cycle Arrest

RKO cells were transfected with pcDNA3.1 (control), pcDNA3.1-p33ING1, orpcDNA3.1-p33ING2. Cells were co-transfected with pEGFP-F Amp (a plasmidcontaining an enhanced green fluorescent protein and an ampicillintransfection marker). The cells were gated by GFP. The GFP-positivecells were considered to be pcDNA3.1, pcDNA3.1-p33ING1, orpcDNA3.1-p33ING2 positive. The propidium iodide signal was used as ameasure for DNA content to determine cell cycle profiles on a FACScanflow cytometer (Becton-Dickinson). See FIG. 3. The percentages of thecells in each cell cycle phage were calculated by the ModFit program(Becton-Dickinson), and the results are as follows:

pcDNA3.1 G₀/G₁ (43.1%), G2M (32.5%), S-phase (24.4%)

pcDNA3.1-p33ING1 G₀/G₁ (67.1%), G₂M (21.7%), S-phase (11.2%)

pcDNA3.1-p33ING2 G₀/G₁ (71.2%), G₂M (19.9%), S-phase (8.9%)

These results indicate that p33ING1 or p33ING2 can induce G₁ cell cyclearrest in cells.

Example VIII p33ING1 and p33ING2 Can Enhance p21/WAF1, BAX, and IGF BP3Promoter Activities in p53 Wild Type Cell Line RKO

The p53 transcriptional transactivities (p21/WAF1, BAX, or IGF BP3) wereexamined with the Dual-Luciferase Reporter Assay System (Promega,E1910). RKO cells were co-transfected with Renilla Luc vector SV 40(internal control) and p53 responsive reporter vectors, WWP-Luc-p21,PGL3-Luc-BAX, or pUHC13-3-Luc-IGF BP3 BOX B. The cells were alsotransfected with pcDNA3.1, pcDNA3.1-p33ING1, or pcDNA3.1-p33ING2. Theresults of the promoter activity according the luciferase assay are asfollows:

p21/WAF1 promoter activity (average +/−SD)

pcDNA3.1 (100+/−9.3)

pcDNA3.1-p33ING1 (190.8+/−15.6)

pcDNA3.1-p33ING2 (199.8+/−29.2)

BAX promoter activity (average +/−SD)

pcDNA3.1 (100+/−6.2)

pcDNA3.1-p33ING1 (237.6+/−15.4)

pcDNA3.1-p33ING2 (347.9+/−28.5)

IGF BP3 promoter activity (average +/−SD)

pcDNA3.1 (100+/−10.9)

pcDNA3.1-p33ING1 (181.8+/−20.6)

pcDNA3.1-p33ING2 (205.0+/−13.1)

The above results indicate that p33ING1 and p33ING2 enhanced p21/WAF1,BAX, and IGF BP3 promoter activities in p53 wild type cell line RKO.

Example IX p33ING1 and p33ING2 Induces Apoptosis

RKO cells were transfected with pcDNA3.1 (control), pcDNA3.1-p33ING1, orpcDNA3.1-p33ING2 expression vector. Cells were co-transfected withpEGFP-F Amp (transfection marker). The cells were fixed 24 hours aftertransfection. The GFP-positive cells were considered to be pcDNA3.1,pcDNA3.1-p33ING1, or pcDNA3.1-p33ING2 positive. Apoptotic change wasdetermined by DAPI staining and TUNEL assay using fluorescentmicroscope. For TUNEL assay, the following kit and materials were used:Fluorescein FragEL DNA Fragmentation Detection Kit (Oncogene ResearchProducts, Cat. #QIA39)+Tetramethyl-rhodamine-5-dUTP (Roche, Cat. #1534378).

The assay results are as follows.

% apoptotic cells/transfected cells (GFP-positive cells)

pcDNA3.1 (control): 15.3+/−1.3 (average +/−SD)

pcDNA3.1-p33ING1: 40.3+/−3.0

pcDNA3.1-p33ING2: 39.3+/−1.7

The above results indicate that expression or overexpression of p33ING1or p33ING2 induced apoptosis in RKO cells at a higher frequency comparedto the control.

10 1 280 PRT Artificial Sequence Description of Artificial Sequencep33ING2 polypeptide sequence 1 Met Leu Gly Gln Gln Gln Gln Gln Leu TyrSer Ser Ala Ala Leu Leu 1 5 10 15 Thr Gly Glu Arg Ser Arg Leu Leu ThrCys Tyr Val Gln Asp Tyr Leu 20 25 30 Glu Cys Val Glu Ser Leu Pro His AspMet Gln Arg Asn Val Ser Val 35 40 45 Leu Arg Glu Leu Asp Asn Lys Tyr GlnGlu Thr Leu Lys Glu Ile Asp 50 55 60 Asp Val Tyr Glu Lys Tyr Lys Lys GluAsp Asp Leu Asn Gln Lys Lys 65 70 75 80 Arg Leu Gln Gln Leu Leu Gln ArgAla Leu Ile Asn Ser Gln Glu Leu 85 90 95 Gly Asp Glu Lys Ile Gln Ile ValThr Gln Met Leu Glu Leu Val Glu 100 105 110 Asn Arg Ala Arg Gln Met GluLeu His Ser Gln Cys Phe Gln Asp Pro 115 120 125 Ala Glu Ser Glu Arg AlaSer Asp Lys Ala Lys Met Asp Ser Ser Gln 130 135 140 Pro Glu Arg Ser SerArg Arg Pro Arg Arg Gln Arg Thr Ser Glu Ser 145 150 155 160 Arg Asp LeuCys His Met Ala Asn Gly Ile Glu Asp Cys Asp Asp Gln 165 170 175 Pro ProLys Glu Lys Lys Ser Lys Ser Ala Lys Lys Lys Lys Arg Ser 180 185 190 LysAla Lys Gln Glu Arg Glu Ala Ser Pro Val Glu Phe Ala Ile Asp 195 200 205Pro Asn Glu Pro Thr Tyr Cys Leu Cys Asn Gln Val Ser Tyr Gly Glu 210 215220 Met Ile Gly Cys Asp Asn Glu Gln Cys Pro Ile Glu Trp Phe His Phe 225230 235 240 Ser Cys Val Ser Leu Thr Tyr Lys Pro Lys Gly Lys Trp Tyr CysPro 245 250 255 Lys Cys Arg Gly Asp Asn Glu Lys Thr Met Asp Lys Ser ThrGlu Lys 260 265 270 Thr Lys Lys Asp Arg Arg Ser Arg 275 280 2 1080 DNAArtificial Sequence Description of Artificial Sequence p33ING2 nucleicacid sequence (GenBank Accession No. AF05053537) 2 gcggccgcgg ccggtgcatgtgcggctgct ggatgcggag gcggcggcga cggcgcggat 60 cggcaggatg ttagggcagcagcagcagca actgtactcg tcggctgcgc tcctgaccgg 120 ggagcggagc cggctgctcacctgctacgt gcaggactac cttgagtgcg tggagtcgct 180 gccccacgac atgcagaggaacgtgtctgt gctgcgagag ctggacaaca aatatcaaga 240 aacgttaaag gaaattgatgatgtctacga aaaatataag aaagaagatg atttaaacca 300 gaagaaacgt ctacagcagcttctccagag agcactaatt aatagtcaag aattgggaga 360 tgaaaaaata cagattgttacacaaatgct cgaattggtg gaaaatcggg caagacaaat 420 ggagttacac tcacagtgtttccaagatcc tgctgaaagt gaacgagcct cagataaagc 480 aaagatggat tccagccaaccagaaagatc ttcaagaaga ccccgcaggc agcggaccag 540 tgaaagccgt gatttatgtcacatggcaaa tgggattgaa gactgtgatg atcagccacc 600 taaagaaaag aaatccaagtcagcaaagaa aaagaaacgc tccaaggcca agcaggaaag 660 ggaagcttca cctgttgagtttgcaataga tcctaatgaa cctacatact gcttatgcaa 720 ccaagtgtct tatggggagatgataggatg tgacaatgaa cagtgtccaa ttgaatggtt 780 tcacttttca tgtgtttcacttacctataa accaaagggg aaatggtatt gcccaaagtg 840 caggggagat aatgagaaaacaatggacaa aagtactgaa aagacaaaaa aggatagaag 900 atcgaggtag taaaggccatccacatttta aagggttatt tgtcttttat ataattcgtt 960 tgctttcaga aaatgttttagggtaaatgc ataagactat gcaataattt ttaatcatta 1020 gtattaatgg tgtattaaaagttgttgtac tttgaaaaaa aaaaaaaaaa aaaaaaaaaa 1080 3 7 PRT ArtificialSequence Description of Artificial Sequence Degenerate primer used toisolate p33ING2 nucleic acids 3 Met Leu Gly Gln Gln Gln Gln 1 5 4 7 PRTArtificial Sequence Description of Artificial Sequence Degenerate primerused to isolate p33ING2 nucleic acid 4 Lys Lys Asp Arg Arg Ser Arg 1 5 520 PRT Artificial Sequence Description of Artificial Sequence peptide7-26 of p33ING2 (KMP1) 5 Gln Gln Leu Tyr Ser Ser Ala Ala Leu Leu Thr GlyGlu Arg Ser Arg 1 5 10 15 Leu Leu Thr Cys 20 6 280 PRT ArtificialSequence Description of Artificial Sequence missense p33ING2 sequence -Arg 153 to Ser 6 Met Leu Gly Gln Gln Gln Gln Gln Leu Tyr Ser Ser Ala AlaLeu Leu 1 5 10 15 Thr Gly Glu Arg Ser Arg Leu Leu Thr Cys Tyr Val GlnAsp Tyr Leu 20 25 30 Glu Cys Val Glu Ser Leu Pro His Asp Met Gln Arg AsnVal Ser Val 35 40 45 Leu Arg Glu Leu Asp Asn Lys Tyr Gln Glu Thr Leu LysGlu Ile Asp 50 55 60 Asp Val Tyr Glu Lys Tyr Lys Lys Glu Asp Asp Leu AsnGln Lys Lys 65 70 75 80 Arg Leu Gln Gln Leu Leu Gln Arg Ala Leu Ile AsnSer Gln Glu Leu 85 90 95 Gly Asp Glu Lys Ile Gln Ile Val Thr Gln Met LeuGlu Leu Val Glu 100 105 110 Asn Arg Ala Arg Gln Met Glu Leu His Ser GlnCys Phe Gln Asp Pro 115 120 125 Ala Glu Ser Glu Arg Ala Ser Asp Lys AlaLys Met Asp Ser Ser Gln 130 135 140 Pro Glu Arg Ser Ser Arg Arg Pro SerArg Gln Arg Thr Ser Glu Ser 145 150 155 160 Arg Asp Leu Cys His Met AlaAsn Gly Ile Glu Asp Cys Asp Asp Gln 165 170 175 Pro Pro Lys Glu Lys LysSer Lys Ser Ala Lys Lys Lys Lys Arg Ser 180 185 190 Lys Ala Lys Gln GluArg Glu Ala Ser Pro Val Glu Phe Ala Ile Asp 195 200 205 Pro Asn Glu ProThr Tyr Cys Leu Cys Asn Gln Val Ser Tyr Gly Glu 210 215 220 Met Ile GlyCys Asp Asn Glu Gln Cys Pro Ile Glu Trp Phe His Phe 225 230 235 240 SerCys Val Ser Leu Thr Tyr Lys Pro Lys Gly Lys Trp Tyr Cys Pro 245 250 255Lys Cys Arg Gly Asp Asn Glu Lys Thr Met Asp Lys Ser Thr Glu Lys 260 265270 Thr Lys Lys Asp Arg Arg Ser Arg 275 280 7 423 DNA Homo sapiens p33ING2 genomic DNA sequence (exon 1/intron) GenBank Accession No.HSING2S1 7 gcggccgcgg ccggtgcatg tgcggctgct ggatgcggag gcggcggcgacggcgcggat 60 cggcaggatg ttagggcagc agcagcagca actgtactcg tcggctgcgctcctgaccgg 120 ggagcggagc cggctgctca cctgctacgt gcaggactac cttgagtgcgtggagtcgct 180 gccccacgac atgcagagga acgtgtctgt gctgcgagag ctggacaacaaatatcaagg 240 taggggccgc ggggctgccg gcctcgggag ccggtggcgg ggagcctgtccgggggagtg 300 ccaccttccc tttctcccgt gacagtctcc ccgagcgcac cgagggtctgccgagcggga 360 ctgggaggac tggagaccgg gttggcggcc ctccgtggcc ccgcggtgggcgagtgaagg 420 aga 423 8 279 PRT Artificial Sequence Description ofArtificial Sequence p33ING1 8 Met Leu Ser Pro Ala Asn Gly Glu Gln LeuHis Leu Val Asn Tyr Val 1 5 10 15 Glu Asp Tyr Leu Asp Ser Ile Glu SerLeu Pro Phe Asp Leu Gln Arg 20 25 30 Asn Val Ser Leu Met Arg Glu Ile AspAla Lys Tyr Gln Glu Ile Leu 35 40 45 Lys Glu Leu Asp Glu Cys Tyr Glu ArgPhe Ser Arg Glu Thr Asp Gly 50 55 60 Ala Gln Lys Arg Arg Met Leu His CysVal Gln Arg Ala Leu Ile Arg 65 70 75 80 Ser Gln Glu Leu Gly Asp Glu LysIle Gln Ile Val Ser Gln Met Val 85 90 95 Glu Leu Val Glu Asn Arg Thr ArgGln Val Asp Ser His Val Glu Leu 100 105 110 Phe Glu Ala Gln Gln Glu LeuGly Asp Thr Ala Gly Asn Ser Gly Lys 115 120 125 Ala Gly Ala Asp Arg ProLys Gly Glu Ala Ala Ala Gln Ala Asp Lys 130 135 140 Pro Asn Ser Lys ArgSer Arg Arg Gln Arg Asn Asn Glu Asn Arg Glu 145 150 155 160 Asn Ala SerSer Asn His Asp His Asp Asp Gly Ala Ser Gly Thr Pro 165 170 175 Lys GluLys Lys Ala Lys Thr Ser Lys Lys Lys Lys Arg Ser Lys Ala 180 185 190 LysAla Glu Arg Glu Ala Ser Pro Ala Asp Leu Pro Ile Asp Pro Asn 195 200 205Glu Pro Thr Tyr Cys Leu Cys Asn Gln Val Ser Tyr Gly Glu Met Ile 210 215220 Gly Cys Asp Asn Asp Glu Cys Pro Ile Glu Trp Phe His Phe Ser Cys 225230 235 240 Val Gly Leu Asn His Lys Pro Lys Gly Lys Trp Tyr Cys Pro LysCys 245 250 255 Arg Gly Glu Asn Glu Lys Thr Met Asp Lys Ala Leu Glu LysSer Lys 260 265 270 Lys Glu Arg Ala Tyr Asn Arg 275 9 279 PRT ArtificialSequence Description of Artificial Sequence peptide 1-17 and C ofp33ING1 (KMP2) 9 Met Leu Ser Pro Ala Asn Gly Glu Gln Leu His Leu Val AsnTyr Val 1 5 10 15 Glu Asp Tyr Leu Asp Ser Ile Glu Ser Leu Pro Phe AspLeu Gln Arg 20 25 30 Asn Val Ser Leu Met Arg Glu Ile Asp Ala Lys Tyr GlnGlu Ile Leu 35 40 45 Lys Glu Leu Asp Glu Cys Tyr Glu Arg Phe Ser Arg GluThr Asp Gly 50 55 60 Ala Gln Lys Arg Arg Met Leu His Cys Val Gln Arg AlaLeu Ile Arg 65 70 75 80 Ser Gln Glu Leu Gly Asp Glu Lys Ile Gln Ile ValSer Gln Met Val 85 90 95 Glu Leu Val Glu Asn Arg Thr Arg Gln Val Asp SerHis Val Glu Leu 100 105 110 Phe Glu Ala Gln Gln Glu Leu Gly Asp Thr AlaGly Asn Ser Gly Lys 115 120 125 Ala Gly Ala Asp Arg Pro Lys Gly Glu AlaAla Ala Gln Ala Asp Lys 130 135 140 Pro Asn Ser Lys Arg Ser Arg Arg GlnArg Asn Asn Glu Asn Arg Glu 145 150 155 160 Asn Ala Ser Ser Asn His AspHis Asp Asp Gly Ala Ser Gly Thr Pro 165 170 175 Lys Glu Lys Lys Ala LysThr Ser Lys Lys Lys Lys Arg Ser Lys Ala 180 185 190 Lys Ala Glu Arg GluAla Ser Pro Ala Asp Leu Pro Ile Asp Pro Asn 195 200 205 Glu Pro Thr TyrCys Leu Cys Asn Gln Val Ser Tyr Gly Glu Met Ile 210 215 220 Gly Cys AspAsn Asp Glu Cys Pro Ile Glu Trp Phe His Phe Ser Cys 225 230 235 240 ValGly Leu Asn His Lys Pro Lys Gly Lys Trp Tyr Cys Pro Lys Cys 245 250 255Arg Gly Glu Asn Glu Lys Thr Met Asp Lys Ala Leu Glu Lys Ser Lys 260 265270 Lys Glu Arg Ala Tyr Asn Arg 275 10 974 DNA Homo sapiens p33ING2genomic DNA sequence (Exon 2/intron) GenBank Accession No. HSING2S2 10ccaaagagga gtatggtttc atggtttgag ttctaatttc aattctgtaa aaaataacta 60ccttggaaat gttgtgtctg ctaacacatg ataacgttct catttttctt ttcctttttt 120tagaaacgtt aaaggaaatt gatgatgtct acgaaaaata taagaaagaa gatgatttaa 180accagaagaa acgtctacag cagcttctcc agagagcact aattaatagt caagaattgg 240gagatgaaaa aatacagatt gttacacaaa tgctcgaatt ggtggaaaat cgggcaagac 300aaatggagtt acactcacag tgtttccaag atcctgctga aagtgaacga gcctcagata 360aagcaaagat ggattccagc caaccagaaa gatcttcaag aagaccccgc aggcagcgga 420ccagtgaaag ccgtgattta tgtcacatgg caaatgggat tgaagactgt gatgatcagc 480cacctaaaga aaagaaatcc aagtcagcaa agaaaaagaa acgctccaag gccaagcagg 540aaagggaagc ttcacctgtt gagtttgcaa tagatcctaa tgaacctaca tactgcttat 600gcaaccaagt gtcttatggg gagatgatag gatgtgacaa tgaacagtgt ccaattgaat 660ggtttcactt ttcatgtgtt tcacttacct ataaaccaaa ggggaaatgg tattgcccaa 720agtgcagggg agataatgag aaaacaatgg acaaaagtac tgaaaagaca aaaaaggata 780gaagatcgag gtagtaaagg ccatccacat tttaaagggt tatttgtctt ttatataatt 840cgtttgcttt cagaaaatgt tttagggtaa atgcataaga ctatgcaata atttttaatc 900attagtatta atggtgtatt aaaagttgtt gtactttgtc tgtgacctta attttctgca 960ctgagttacc aaat 974

What is claimed is:
 1. An isolated nucleic acid encoding a tumorsuppressor polypeptide p33ING2 that has at least 90% identity to anucleic acid comprising a nucleotide sequence of SEQ ID NO:2, andwherein the p33ING2 polypeptide inhibits cell growth.
 2. The isolatednucleic acid of claim 1, wherein the nucleic acid encodes a polypeptidecomprising an amino acid sequence of SEQ ID NO:1.
 3. The isolatednucleic acid sequence of claim 1, wherein the nucleic acid comprises anucleotide sequence of SEQ ID NO:2.
 4. The isolated nucleic acid ofclaim 1, wherein the nucleic acid is from a human.
 5. The isolatednucleic acid of claim 1, wherein the nucleic acid encodes a polypeptidehaving a molecular weight of about 28 kDa to about 38 kDa.
 6. Anexpression vector comprising the nucleic acid of claim
 1. 7. An isolatedhost cell transfected with the vector of claim
 6. 8. The isolatednucleic acid of claim 1, wherein the nucleic acid has at least 95%identity to a nucleic acid comprising a nucleotide sequence of SEQ IDNO:2.